Murine anti-NY-ESO-1 T cell receptors

ABSTRACT

The invention provides an isolated or purified T cell receptor (TCR) having antigenic specificity for NY-ESO-1. Also provided are related polypeptides, proteins, nucleic acids, recombinant expression vectors, isolated host cells, populations of cells, antibodies, or antigen binding portions thereof, and pharmaceutical compositions. The invention further provides a method of detecting the presence of cancer in a mammal and a method of treating or preventing cancer in a mammal using the inventive TCRs or related materials.

CROSS REFERENCE TO RELATED APPLICATION

This patent application is a continuation of U.S. patent applicationSer. No. 14/401,893, which is the U.S. National Phase of InternationalPatent Application No. PCT/US2013/042162, filed May 22, 2013, whichclaims the benefit of U.S. Provisional Patent Application No.61/650,020, filed May 22, 2012, each of which is incorporated byreference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under project numberZIABC010984 by the National Institutes of Health, National CancerInstitute. The Government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 23,552 Byte ASCII (Text) file named“726472_ST25.TXT,” dated Sep. 13, 2016.

BACKGROUND OF THE INVENTION

Adoptive cell therapy can be an effective treatment for cancer in somepatients. However, obstacles to the overall success of adoptive celltherapy still exist. For example, only 50% of melanoma tumor samples maygenerate tumor reactive T-cells. Generating tumor-reactive T-cells fromnon-melanoma cancers can also be difficult. Moreover, many patients maynot have a tumor that is amenable to surgical resection. Accordingly,there is a need for T-cell receptors for use in treating patients withcancer.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated or purified T-cell receptor (TCR)having antigenic specificity for NY-ESO-1 and comprising a murinevariable region. The invention also provides related polypeptides andproteins, as well as related nucleic acids, recombinant expressionvectors, host cells, and populations of cells. Further provided by theinvention are antibodies, or an antigen binding portion thereof, andpharmaceutical compositions relating to the TCRs of the invention.

Methods of detecting the presence of cancer in a mammal and methods oftreating or preventing cancer in a mammal are further provided by theinvention. The inventive method of detecting the presence of cancer in amammal comprises (i) contacting a sample comprising cells of the cancerwith any of the inventive TCRs, polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of host cells,or antibodies, or antigen binding portions thereof, described herein,thereby forming a complex, and (ii) detecting the complex, whereindetection of the complex is indicative of the presence of cancer in themammal.

The inventive method of treating or preventing cancer in a mammalcomprises administering to the mammal any of the TCRs, polypeptides, orproteins described herein, any nucleic acid or recombinant expressionvector comprising a nucleotide sequence encoding any of the TCRs,polypeptides, proteins described herein, or any host cell or populationof host cells comprising a recombinant vector which encodes any of theTCRs, polypeptides, or proteins described herein, in an amount effectiveto treat or prevent cancer in the mammal.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1A and 1B are graphs showing interferon (IFN)-γ secretion by humanCD8+ (FIG. 1A) or CD4+ (FIG. 1B) T cells transfected with a murineanti-NY-ESO-1 TCR (shaded circles) or a human anti-NY-ESO-1 TCR(unshaded circles) upon co-culture with dendritic cells pulsed withvarious concentrations of NY-ESO-1₁₅₇₋₁₆₅.

FIGS. 2A and 2B are graphs showing IFN-γ secretion by human CD8+ (FIG.2A) or CD4+ (FIG. 2B) T cells transfected with a murine anti-NY-ESO-1TCR (shaded bars) or a human anti-NY-ESO-1 TCR (unshaded bars) culturedalone (media) or co-cultured with T2 cells pulsed with control peptide,T2 cells pulsed with NY-ESO-1₁₅₇₋₁₆₅ peptide, or one of various tumorcell lines 888mel (NY-ESO-1⁻), Sk23mel (NY-ESO-1⁻), COA-A2-CEA(NY-ESO-1⁻), A375mel (NY-ESO-1⁺), 1363mel (NY-ESO-1⁺), or COS-A2-ESO(NY-ESO-1⁺).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated or purified T-cell receptor (TCR)having antigenic specificity for NY-ESO-1 and comprising a murinevariable region. NY-ESO-1 is a cancer testis antigen (CTA), which isexpressed only in tumor cells and non-MHC expressing germ cells of thetestis and placenta. NY-ESO-1 is expressed in a variety of human cancersincluding, but not limited to, melanoma, breast cancer, lung cancer,prostate cancer, thyroid cancer, ovarian cancer, and synovial cellsarcoma. The NY-ESO-1 protein may comprise, consist, or consistessentially of, SEQ ID NO: 1.

The TCR may have antigenic specificity for any NY-ESO-1 protein,polypeptide or peptide. In an embodiment of the invention, the TCR hasantigenic specificity for a NY-ESO-1 protein comprising, consisting of,or consisting essentially of, SEQ ID NO: 1. In a preferred embodiment ofthe invention, the TCR has antigenic specificity for a NY-ESO-1 157-165peptide comprising, consisting of, or consisting essentially of,SLLMWITQC (SEQ ID NO: 2).

The phrase “having antigenic specificity” as used herein means that theTCR can specifically bind to and immunologically recognize NY-ESO-1,such that binding of the TCR to NY-ESO-1 elicits an immune response.

In an embodiment of the invention, the inventive TCRs are able torecognize NY-ESO-1 in a major histocompatibility complex (MHC) classI-dependent manner. By “MHC class I-dependent manner” as used hereinmeans that the TCR elicits an immune response upon binding to NY-ESO-1within the context of an MHC class I molecule. The MHC class I moleculecan be any MHC class I molecule known in the art, e.g., HLA-A molecules.In an embodiment of the invention, the MHC class I molecule is an HLA-A2molecule.

In an embodiment of the invention, the inventive TCRs comprise a murinevariable region. The inventive TCRs may further comprise a constantregion derived from any suitable species such as, e.g., human or mouse.Preferably, the inventive TCRs further comprise a murine constantregion. In an especially preferred embodiment, the inventive TCRs aremurine TCRs comprising both a murine variable region and a murineconstant region.

As used herein, the term “murine,” when referring to a TCR or anycomponent of a TCR described herein (e.g., complementarity determiningregion (CDR), variable region, constant region, alpha chain, and/or betachain), means a TCR (or component thereof) which is derived from amouse, i.e., a TCR (or component thereof) that originated from or was,at one time, expressed by a mouse T cell. Desirably, the TCR (orcomponent thereof) is expressed on the surface of a human host cell.

The TCRs of the invention provide many advantages, including when usedfor adoptive cell transfer. For example, without being bound by aparticular theory or mechanism, it is believed that because NY-ESO-1 isexpressed by cells of multiple cancer types, the inventive TCRsadvantageously provide the ability to destroy cells of multiple types ofcancer and, accordingly, treat or prevent multiple types of cancer.Additionally, without being bound to a particular theory or mechanism,it is believed that because NY-ESO-1 is a cancer testis antigen that isexpressed only in tumor cells and non-MHC expressing germ cells of thetestis and placenta, the inventive TCRs advantageously target thedestruction of cancer cells while minimizing or eliminating thedestruction of normal, non-cancerous cells, thereby reducing, forexample, minimizing or eliminating, toxicity. It is also believed thatmurine TCRs may provide increased expression (e.g., higher numbers ofTCRs) on the surface of a human host cell and/or increased functionality(as measured by, e.g., cytokine release and cytotoxicity) as compared toa human TCR. Without being bound to a particular theory of mechanism, itis believed that the improved expression and/or functionality resultsfrom a reduction in the mixing of endogenous and exogenous (transduced)TCR chains in the host cell. Accordingly, it is believed that murineTCRs can replace endogenous TCRs on the surface of a human host cellmore effectively than an exogenous human TCR. It is also believed thatmurine TCRs provide improved pairing of TCR chains and/or improvedinteractions with the CD3 complex of the human host cell as compared toexogenous human TCRs expressed by a human host cell.

An embodiment of the invention provides a TCR comprising twopolypeptides (i.e., polypeptide chains), such as an α chain of a TCR, aβ chain of a TCR, a γ chain of a TCR, a δ chain of a TCR, or acombination thereof. The polypeptides of the inventive TCR can compriseany amino acid sequence, provided that the TCR has antigenic specificityfor NY-ESO-1 and comprises a murine variable region.

In a preferred embodiment of the invention, the TCR comprises twopolypeptide chains, each of which comprises a variable region comprisinga complementarity determining region (CDR) 1, a CDR2, and a CDR3 of aTCR. Preferably, the first polypeptide chain comprises a CDR1 comprisingthe amino acid sequence of SEQ ID NO: 3 (CDR1 of α chain), a CDR2comprising the amino acid sequence of SEQ ID NO: 4 (CDR2 of α chain),and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5 (CDR3 of αchain), and the second polypeptide chain comprises a CDR1 comprising theamino acid sequence of SEQ ID NO: 6 (CDR1 of β chain), a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 7 (CDR2 of β chain), and a CDR3comprising the amino acid sequence of SEQ ID NO: 8 (CDR3 of β chain). Inthis regard, the inventive TCR can comprise the amino acid sequencesselected from the group consisting of SEQ ID NOs: 3-5, 6-8, and 3-8.Preferably the TCR comprises the amino acid sequences of SEQ ID NOs:3-8.

Alternatively or additionally, the TCR can comprise an amino acidsequence of a variable region of a TCR comprising the CDRs set forthabove. In this regard, the TCR can comprise the amino acid sequence ofSEQ ID NO: 9 (the variable region of an α chain) or 10 (the variableregion of a β chain), or both SEQ ID NOs: 9 and 10. Preferably, theinventive TCR comprises the amino acid sequences of SEQ ID NOs: 9 and10.

Alternatively or additionally, the TCR can comprise an α chain of a TCRand a β chain of a TCR. Each of the α chain and β chain of the inventiveTCR can independently comprise any amino acid sequence. Preferably, theα chain comprises the variable region of an α chain as set forth above.In this regard, the inventive TCR can comprise the amino acid sequenceof SEQ ID NO: 11. An inventive TCR of this type can be paired with any βchain of a TCR. Preferably, the β chain of the inventive TCR comprisesthe variable region of β chain as set forth above. In this regard, theinventive TCR can comprise the amino acid sequence of SEQ ID NO: 12. Theinventive TCR, therefore, can comprise the amino acid sequence of SEQ IDNO: 11 or 12, or both SEQ ID NOs: 11 and 12. Preferably, the inventiveTCR comprises the amino acid sequences of SEQ ID NOs: 11 and 12.

Also provided by the invention is an isolated or purified polypeptidecomprising a functional portion of any of the TCRs described herein. Theterm “polypeptide” as used herein includes oligopeptides and refers to asingle chain of amino acids connected by one or more peptide bonds.

With respect to the inventive polypeptides, the functional portion canbe any portion comprising contiguous amino acids of the TCR of which itis a part, provided that the functional portion specifically binds toNY-ESO-1. The term “functional portion” when used in reference to a TCRrefers to any part or fragment of the TCR of the invention, which partor fragment retains the biological activity of the TCR of which it is apart (the parent TCR). Functional portions encompass, for example, thoseparts of a TCR that retain the ability to specifically bind to NY-ESO-1,or detect, treat, or prevent cancer, to a similar extent, the sameextent, or to a higher extent, as the parent TCR. In reference to theparent TCR, the functional portion can comprise, for instance, about10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent TCR.

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent TCR.Desirably, the additional amino acids do not interfere with thebiological function of the functional portion, e.g., specificallybinding to NY-ESO-1, having the ability to detect cancer, treat orprevent cancer, etc. More desirably, the additional amino acids enhancethe biological activity, as compared to the biological activity of theparent TCR.

The polypeptide can comprise a functional portion of either or both ofthe α and α chains of the TCRs of the invention, such as a functionalportion comprising one or more of CDR1, CDR2, and CDR3 of the variableregion(s) of the α chain and/or β chain of a TCR of the invention. Inthis regard, the polypeptide can comprise a functional portioncomprising the amino acid sequence of SEQ ID NO: 3 (CDR1 of α chain), 4(CDR2 of α chain), 5 (CDR3 of α chain), 6 (CDR1 of β chain), 7 (CDR2 ofβ chain), 8 (CDR3 of β chain), or a combination thereof. Preferably, theinventive polypeptide comprises a functional portion comprising SEQ IDNOs: 3-5, 6-8, or all of SEQ ID NOs: 3-8. More preferably, thepolypeptide comprises a functional portion comprising the amino acidsequences of SEQ ID NOs: 3-8.

Alternatively or additionally, the inventive polypeptide can comprise,for instance, the variable region of the inventive TCR comprising acombination of the CDR regions set forth above. In this regard, the TCRcan comprise the amino acid sequence of SEQ ID NO: 9 (the variableregion of an α chain) or 10 (the variable region of a β chain), or bothSEQ ID NOs: 9 and 10. Preferably, the polypeptide comprises the aminoacid sequences of SEQ ID NOs: 9 and 10.

Alternatively or additionally, the inventive polypeptide can comprisethe entire length of an α or β chain of one of the TCRs describedherein. In this regard, the inventive polypeptide can comprise an aminoacid sequence of SEQ ID NO: 11 or 12. Alternatively, the polypeptide ofthe invention can comprise both chains of the TCRs described herein. Forexample, the inventive polypeptide can comprise both amino acidsequences of SEQ ID NOs: 11 and 12.

The invention further provides an isolated or purified proteincomprising at least one of the polypeptides described herein. By“protein” is meant a molecule comprising one or more polypeptide chains.

The protein of the invention can comprise a first polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 9 and a secondpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 10.The protein of the invention can, for example, comprise a firstpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 11and a second polypeptide chain comprising the amino acid sequence of SEQID NO: 12. In this instance, the protein of the invention can be a TCR.Alternatively, if, for example, the protein comprises a singlepolypeptide chain comprising SEQ ID NO: 11 and SEQ ID NO: 12, or if thefirst and/or second polypeptide chain(s) of the protein furthercomprise(s) other amino acid sequences, e.g., an amino acid sequenceencoding an immunoglobulin or a portion thereof, then the inventiveprotein can be a fusion protein. In this regard, the invention alsoprovides a fusion protein comprising at least one of the inventivepolypeptides described herein along with at least one other polypeptide.The other polypeptide can exist as a separate polypeptide of the fusionprotein, or can exist as a polypeptide, which is expressed in frame (intandem) with one of the inventive polypeptides described herein. Theother polypeptide can encode any peptidic or proteinaceous molecule, ora portion thereof, including, but not limited to an immunoglobulin, CD3,CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1 b, CD1c,CD1d, etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. See, for instance, Choi et al., Mol.Biotechnol. 31: 193-202 (2005).

In some embodiments of the invention, the TCRs, polypeptides, andproteins of the invention (including functional portions and functionalvariants) may be expressed as a single protein comprising a linkerpeptide linking the α chain and the β chain. Any linker peptide suitablefor linking the α chain and the β chain may be used in the TCRs,polypeptides, and proteins (including functional portions and functionalvariants) of the invention. In an embodiment of the invention, thelinker peptide is a picornavirus 2A peptide. In this regard, the TCRs,polypeptides, and proteins of the invention (including functionalportions and functional variants) may further comprise a linker peptidecomprising an amino acid sequence comprising SEQ ID NO: 13. The linkerpeptide may advantageously facilitate the expression of a recombinantTCR, polypeptide, and/or protein in a host cell. Upon expression of theconstruct including the linker peptide by a host cell, the linkerpeptide may be cleaved, resulting in separated a and β chains.

The protein of the invention can be a recombinant antibody comprising atleast one of the inventive polypeptides described herein. As usedherein, “recombinant antibody” refers to a recombinant (e.g.,genetically engineered) protein comprising at least one of thepolypeptides of the invention and a polypeptide chain of an antibody, ora portion thereof. The polypeptide of an antibody, or portion thereof,can be a heavy chain, a light chain, a variable or constant region of aheavy or light chain, a single chain variable fragment (scFv), or an Fc,Fab, or F(ab)₂′ fragment of an antibody, etc. The polypeptide chain ofan antibody, or portion thereof, can exist as a separate polypeptide ofthe recombinant antibody. Alternatively, the polypeptide chain of anantibody, or portion thereof, can exist as a polypeptide, which isexpressed in frame (in tandem) with the polypeptide of the invention.The polypeptide of an antibody, or portion thereof, can be a polypeptideof any antibody or any antibody fragment, including any of theantibodies and antibody fragments described herein.

Included in the scope of the invention are functional variants of theinventive TCRs, polypeptides, and proteins described herein. The term“functional variant” as used herein refers to a TCR, polypeptide, orprotein having substantial or significant sequence identity orsimilarity to a parent TCR, polypeptide, or protein, which functionalvariant retains the biological activity of the TCR, polypeptide, orprotein of which it is a variant. Functional variants encompass, forexample, those variants of the TCR, polypeptide, or protein describedherein (the parent TCR, polypeptide, or protein) that retain the abilityto specifically bind to NY-ESO-1 to a similar extent, the same extent,or to a higher extent, as the parent TCR, polypeptide, or protein. Inreference to the parent TCR, polypeptide, or protein, the functionalvariant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical in aminoacid sequence to the parent TCR, polypeptide, or protein.

The functional variant can, for example, comprise the amino acidsequence of the parent TCR, polypeptide, or protein with at least oneconservative amino acid substitution. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the samechemical or physical properties. For instance, the conservative aminoacid substitution can be an acidic amino acid substituted for anotheracidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar sidechain substituted for another amino acid with a nonpolar side chain(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basicamino acid substituted for another basic amino acid (Lys, Arg, etc.), anamino acid with a polar side chain substituted for another amino acidwith a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent TCR, polypeptide, or protein with atleast one non-conservative amino acid substitution. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the parent TCR, polypeptide, or protein.

The TCR, polypeptide, or protein can consist essentially of thespecified amino acid sequence or sequences described herein, such thatother components of the functional variant, e.g., other amino acids, donot materially change the biological activity of the functional variant.In this regard, the inventive TCR, polypeptide, or protein can, forexample, consist essentially of the amino acid sequence of SEQ ID NO: 11or 12, or both SEQ ID NOs: 11 and 12. Also, for instance, the inventiveTCRs, polypeptides, or proteins can consist essentially of the aminoacid sequence(s) of SEQ ID NO: 9 or 10, or both SEQ ID NOs: 9 and 10.Furthermore, the inventive TCRs, polypeptides, or proteins can consistessentially of the amino acid sequence of SEQ ID NO: 3 (CDR1 of αchain), 4 (CDR2 of α chain), 5 (CDR3 of α chain), 6 (CDR1 of β chain), 7(CDR2 of β chain), 8 (CDR3 of β chain), or any combination thereof,e.g., SEQ ID NOs: 3-5, 6-8, or 3-8.

The TCRs, polypeptides, and proteins of the invention (includingfunctional portions and functional variants) can be of any length, i.e.,can comprise any number of amino acids, provided that the TCRs,polypeptides, or proteins (or functional portions or functional variantsthereof) retain their biological activity, e.g., the ability tospecifically bind to NY-ESO-1, detect cancer in a mammal, or treat orprevent cancer in a mammal, etc. For example, the polypeptide can be 50to 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200,300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.In this regard, the polypeptides of the invention also includeoligopeptides.

The TCRs, polypeptides, and proteins of the invention (includingfunctional portions and functional variants) of the invention cancomprise synthetic amino acids in place of one or morenaturally-occurring amino acids. Such synthetic amino acids are known inthe art, and include, for example, aminocyclohexane carboxylic acid,norleucine, α-amino n-decanoic acid, homoserine,5-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine,phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine,indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, aminomalonic acid, aminomalonic acid monoamide,N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine,ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptane carboxylic acid,α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid,α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

The TCRs, polypeptides, and proteins of the invention (includingfunctional portions and functional variants) can be glycosylated,amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclizedvia, e.g., a disulfide bridge, or converted into an acid addition saltand/or optionally dimerized or polymerized, or conjugated.

When the TCRs, polypeptides, and proteins of the invention (includingfunctional portions and functional variants) are in the form of a salt,preferably, the polypeptides are in the form of a pharmaceuticallyacceptable salt. Suitable pharmaceutically acceptable acid additionsalts include those derived from mineral acids, such as hydrochloric,hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids,and organic acids, such as tartaric, acetic, citric, malic, lactic,fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids,for example, p-toluenesulphonic acid.

The TCR, polypeptide, and/or protein of the invention (includingfunctional portions and functional variants thereof) can be obtained bymethods known in the art. Suitable methods of de novo synthesizingpolypeptides and proteins are described in references, such as Chan etal., Fmoc Solid Phase Peptide Synthesis, Oxford University Press,Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed.Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwoood etal., Oxford University Press, Oxford, United Kingdom, 2000; and U.S.Pat. No. 5,449,752. Also, polypeptides and proteins can be recombinantlyproduced using the nucleic acids described herein using standardrecombinant methods. See, for instance, Sambrook et al., MolecularCloning: A Laboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Further, some of the TCRs, polypeptides, and proteins of theinvention (including functional portions and functional variantsthereof) can be isolated and/or purified from a source, such as a plant,a bacterium, an insect, a mammal, e.g., a mouse, a human, etc. Methodsof isolation and purification are well-known in the art. Alternatively,the TCRs, polypeptides, and/or proteins described herein (includingfunctional portions and functional variants thereof) can be commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the inventive TCRs, polypeptides,and proteins can be synthetic, recombinant, isolated, and/or purified.

Included in the scope of the invention are conjugates, e.g.,bioconjugates, comprising any of the inventive TCRs, polypeptides, orproteins (including any of the functional portions or variants thereof),nucleic acids, recombinant expression vectors, host cells, populationsof host cells, or antibodies, or antigen binding portions thereof.Conjugates, as well as methods of synthesizing conjugates in general,are known in the art (See, for instance, Hudecz, F., Methods Mol. Biol.298: 209-223 (2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415(2005)).

Further provided by the invention is a nucleic acid comprising anucleotide sequence encoding any of the TCRs, polypeptides, or proteinsdescribed herein (including functional portions and functional variantsthereof).

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. It is generally preferred that thenucleic acid does not comprise any insertions, deletions, inversions,and/or substitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., supra, and Ausubel et al., supra. For example,a nucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any nucleotide sequence which encodes anyof the inventive TCRs, polypeptides, or proteins, or functional portionsor functional variants thereof. For example, the nucleic acid cancomprise a nucleotide sequence comprising, consisting of, or consistingessentially of SEQ ID NO: 19 (wild-type α chain) or SEQ ID NO: 20(wild-type β chain) or both SEQ ID NOs: 19 and 20.

In some embodiments, the nucleotide sequence may be codon-optimized.Without being bound to a particular theory or mechanism, it is believedthat codon optimization of the nucleotide sequence increases thetranslation efficiency of the mRNA transcripts. Codon optimization ofthe nucleotide sequence may involve substituting a native codon foranother codon that encodes the same amino acid, but can be translated bytRNA that is more readily available within a cell, thus increasingtranslation efficiency. Optimization of the nucleotide sequence may alsoreduce secondary mRNA structures that would interfere with translation,thus increasing translation efficiency. In an embodiment of theinvention, the codon-optimized nucleotide sequence may comprise,consist, or consist essentially of SEQ ID NO: 15 (codon-optimized αchain), SEQ ID NO: 16 (codon-optimized 13 chain), SEQ ID NO: 21(codon-optimized variable region of α chain), SEQ ID NO: 22(codon-optimized variable region of β chain), both SEQ ID NOs: 15 and16, both SEQ ID NOs: 21 and 22, both SEQ ID NOs: 15 and 20, or both SEQID NOs: 16 and 19.

In an embodiment of the invention, the nucleotide sequence encoding theTCRs, polypeptides, and proteins of the invention (including functionalportions and functional variants thereof) may further comprise anucleotide sequence encoding any of the linker peptides described hereinwith respect to other aspects of the invention. In an embodiment of theinvention, the linker peptide may be encoded by a nucleotide sequencecomprising SEQ ID NO: 14.

The invention also provides a nucleic acid comprising a nucleotidesequence that is at least about 70% or more, e.g., about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to any of the nucleic acidsdescribed herein.

The nucleotide sequence alternatively can comprise a nucleotide sequencewhich is degenerate to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, both SEQ ID NOs: 15 and 16,both SEQ ID NOs: 19 and 20, both SEQ ID NOs: 21 and 22, both SEQ ID NOs:15 and 20, or both SEQ ID NOs: 16 and 19. Preferably, the nucleic acidcomprises a nucleotide sequence comprising SEQ ID NO: 15, 16, 19, 20,21, or 22, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21and 22, SEQ ID NOs: 15 and 20, or SEQ ID NOs: 16 and 19, or a nucleotidesequence which is degenerate thereto.

The invention also provides an isolated or purified nucleic acidcomprising a nucleotide sequence which is complementary to thenucleotide sequence of any of the nucleic acids described herein or anucleotide sequence which hybridizes under stringent conditions to thenucleotide sequence of any of the nucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditionspreferably hybridizes under high stringency conditions. By “highstringency conditions” is meant that the nucleotide sequencespecifically hybridizes to a target sequence (the nucleotide sequence ofany of the nucleic acids described herein) in an amount that isdetectably stronger than non-specific hybridization. High stringencyconditions include conditions which would distinguish a polynucleotidewith an exact complementary sequence, or one containing only a fewscattered mismatches from a random sequence that happened to have a fewsmall regions (e.g., 3-10 bases) that matched the nucleotide sequence.Such small regions of complementarity are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive TCRs. It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

The nucleic acids of the invention can be incorporated into arecombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the nucleic acids ofthe invention. For purposes herein, the term “recombinant expressionvector” means a genetically-modified oligonucleotide or polynucleotideconstruct that permits the expression of an mRNA, protein, polypeptide,or peptide by a host cell, when the construct comprises a nucleotidesequence encoding the mRNA, protein, polypeptide, or peptide, and thevector is contacted with the cell under conditions sufficient to havethe mRNA, protein, polypeptide, or peptide expressed within the cell.The vectors of the invention are not naturally-occurring as a whole.However, parts of the vectors can be naturally-occurring. The inventiverecombinant expression vectors can comprise any type of nucleotides,including, but not limited to DNA and RNA, which can be single-strandedor double-stranded, synthesized or obtained in part from naturalsources, and which can contain natural, non-natural or alterednucleotides. The recombinant expression vectors can comprisenaturally-occurring, non-naturally-occurring internucleotide linkages,or both types of linkages. Preferably, the non-naturally occurring oraltered nucleotides or internucleotide linkages do not hinder thetranscription or replication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host cell. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used.Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-C1, pMAM and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector or a lentiviral vector.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColE1, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host cell (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected host cells.Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic hostto provide prototrophy, and the like. Suitable marker genes for theinventive expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding the TCR,polypeptide, or protein (including functional portions and functionalvariants thereof), or to the nucleotide sequence which is complementaryto or which hybridizes to the nucleotide sequence encoding the TCR,polypeptide, or protein. The selection of promoters, e.g., strong, weak,inducible, tissue-specific and developmental-specific, is within theordinary skill of the artisan. Similarly, the combining of a nucleotidesequence with a promoter is also within the skill of the artisan. Thepromoter can be a non-viral promoter or a viral promoter, e.g., acytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and apromoter found in the long-terminal repeat of the murine stem cellvirus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression. Further, the recombinant expression vectorscan be made to include a suicide gene.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene can be a genethat confers sensitivity to an agent, e.g., a drug, upon the cell inwhich the gene is expressed, and causes the cell to die when the cell iscontacted with or exposed to the agent. Suicide genes are known in theart (see, for example, Suicide Gene Therapy: Methods and Reviews,Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeuticsat the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press,2004) and include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase,and nitroreductase.

The inventive recombinant expression vectors may comprise a nucleotidesequence encoding all or a portion of the alpha chain positioned 5′ ofthe nucleotide sequence encoding all or a portion of the beta chain. Inthis regard, an embodiment of the invention provides a recombinantexpression vector comprising a nucleotide sequence encoding a CDR 1α,CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β, and the nucleotide sequenceencoding the CDR1α, CDR2α, and CDR3α is 5′ of the nucleotide sequenceencoding the CDR1β, CDR2β, and CDR3β. Likewise, the nucleotide sequenceencoding the CDR1β, CDR2β, and CDR3β may be 3′ of the nucleotidesequence encoding the CDR1α, CDR2α, and CDR3α. In another embodiment ofthe invention, the recombinant expression vector comprises a nucleotidesequence encoding a variable region of the alpha chain and a variableregion of the beta chain, and the nucleotide sequence encoding thevariable region of the alpha chain is 5′ of the nucleotide sequenceencoding the variable region of the beta chain. Likewise, the nucleotidesequence encoding the variable region of the beta chain may be 3′ of thenucleotide sequence encoding the variable region of the alpha chain. Instill another embodiment of the invention, the recombinant expressionvector comprises a nucleotide sequence encoding an alpha chain and abeta chain, and the nucleotide sequence encoding the alpha chain is 5′of the nucleotide sequence encoding the beta chain. Likewise, thenucleotide sequence encoding the beta chain may be 3′ of the nucleotidesequence encoding the alpha chain. The recombinant expression vectorcomprising a nucleotide sequence encoding all or a portion of the alphachain positioned 5′ of the nucleotide sequence encoding all or a portionof the beta chain may comprise SEQ ID NO: 17.

The inventive recombinant expression vectors may comprise a nucleotidesequence encoding all or a portion of the alpha chain positioned 3′ ofthe nucleotide sequence encoding all or a portion of the beta chain.Without being bound by a particular theory or mechanism, it is believedthat a TCR, polypeptide, or protein (or functional portion or variantthereof) encoded by a recombinant expression vector in which thenucleotide sequence encoding all or a portion of the alpha chain ispositioned 3′ of the nucleotide sequence encoding all or a portion ofthe beta chain provides improved functionality and antigen recognitionas compared to a TCR, polypeptide, or protein (or functional portion orfunctional variant thereof) encoded by a recombinant expression vectorin which the nucleotide sequence encoding all or a portion of the alphachain is positioned 5′ of the nucleotide sequence encoding all or aportion of the beta chain. In this regard, an embodiment of theinvention provides a recombinant expression vector comprising anucleotide sequence encoding a CDR 1α, CDR2α, CDR3α, CDR1β, CDR2β, andCDR3β, and the nucleotide sequence encoding the CDR1α, CDR2α, and CDR3αis 3′ of the nucleotide sequence encoding the CDR1β, CDR2β, and CDR3β.Likewise, the nucleotide sequence encoding the CDR1β, CDR2β, and CDR3βmay be 5′ of the nucleotide sequence encoding the CDR1α, CDR2α, andCDR3α. In another embodiment of the invention, the recombinantexpression vector comprises a nucleotide sequence encoding a variableregion of the alpha chain and a variable region of the beta chain, andthe nucleotide sequence encoding the variable region of the alpha chainis 3′ of the nucleotide sequence encoding the variable region of thebeta chain. Likewise, the nucleotide sequence encoding the variableregion of the beta chain may be 5′ of the nucleotide sequence encodingthe variable region of the alpha chain. In still another embodiment ofthe invention, the recombinant expression vector comprises a nucleotidesequence encoding an alpha chain and a beta chain, and the nucleotidesequence encoding the alpha chain is 3′ of the nucleotide sequenceencoding the beta chain. Likewise, the nucleotide sequence encoding thebeta chain may be 5′ of the nucleotide sequence encoding the alphachain. The recombinant expression vector comprising a nucleotidesequence encoding all or a portion of the alpha chain positioned 3′ ofthe nucleotide sequence encoding all or a portion of the beta chain maycomprise SEQ ID NO: 18.

In an embodiment of the invention, the recombinant expression vector maycomprise a DNA tag. The DNA tag may distinguish the recombinantexpression vector from another vector encoding the same proteinsequence. The DNA tag may not be included within the nucleotide sequenceencoding the inventive TCR (including functional portions and functionalvariants thereof), polypeptide, or protein and, therefore, may notaffect its expression. Recombinant expression vectors including the DNAtag make it possible to put the same nucleotide sequence into severaldifferent cell populations and subsequently distinguish between thosepopulations based on which vector they contain.

The invention further provides a host cell comprising any of therecombinant expression vectors described herein. As used herein, theterm “host cell” refers to any type of cell that can contain theinventive recombinant expression vector. The host cell can be aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5α E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell is preferably aprokaryotic cell, e.g., a DH5α cell. For purposes of producing arecombinant TCR, polypeptide, or protein, the host cell is preferably amammalian cell. Most preferably, the host cell is a human cell. Whilethe host cell can be of any cell type, can originate from any type oftissue, and can be of any developmental stage, the host cell may be aperipheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell(PBMC). Preferably, the host cell may be a T cell.

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. The T cell may be a human T cell. The T cell may be a Tcell isolated from a human. The T cell can be any type of T cell and canbe of any developmental stage, including but not limited to, CD4/CD8⁺double positive T cells, CD4⁺ helper T cells, e.g., Th₁ and Th₂ cells,CD8⁺ T cells, cytotoxic T cells, tumor infiltrating lymphocyte cells,memory T cells, nave T cells, and the like. Preferably, the T cell maybe a CD8⁺ T cell or a CD4⁺ T cell.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells can be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell (e.g., a T cell), which does not compriseany of the recombinant expression vectors, or a cell other than a Tcell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, ahepatocyte, an endothelial cell, an epithelial cells, a muscle cell, abrain cell, etc. Alternatively, the population of cells can be asubstantially homogeneous population, in which the population comprisesmainly of host cells (e.g., consisting essentially of) comprising therecombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

The invention further provides an antibody, or antigen binding portionthereof, which specifically binds to a functional portion of any of theTCRs described herein. Preferably, the functional portion specificallybinds to NY-ESO-1, e.g., the functional portion comprising the aminoacid sequence SEQ ID NO: 3 (CDR1 of α chain), 4 (CDR2 of α chain), 5(CDR3 of α chain), 6 (CDR1 of β chain), 7 (CDR2 of β chain), 8 (CDR3 ofβ chain), SEQ ID NO: 9, SEQ ID NO: 10, or a combination thereof, e.g.,3-5, 6-8, 3-8, or 9-10. More preferably, the functional portioncomprises the amino acid sequences of SEQ ID NOs: 3-8. In a preferredembodiment, the antibody, or antigen binding portion thereof, binds toan epitope which is formed by all 6 CDRs (CDR1-3 of the alpha chain andCDR1-3 of the beta chain). The antibody can be any type ofimmunoglobulin that is known in the art. For instance, the antibody canbe of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody canbe monoclonal or polyclonal. The antibody can be a naturally-occurringantibody, e.g., an antibody isolated and/or purified from a mammal,e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.Alternatively, the antibody can be a genetically-engineered antibody,e.g., a humanized antibody or a chimeric antibody. The antibody can bein monomeric or polymeric form. Also, the antibody can have any level ofaffinity or avidity for the functional portion of the inventive TCR.Desirably, the antibody is specific for the functional portion of theinventive TCR, such that there is minimal cross-reaction with otherpeptides or proteins.

Methods of testing antibodies for the ability to bind to any functionalportion of the inventive TCR are known in the art and include anyantibody-antigen binding assay, such as, for example, radioimmunoassay(RIA), ELISA, Western blot, immunoprecipitation, and competitiveinhibition assays (see, e.g., Janeway et al., infra).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Köhler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 5^(th) to Garland Publishing, New York, N.Y.(2001)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352).

Phage display furthermore can be used to generate the antibody of theinvention. In this regard, phage libraries encoding antigen-bindingvariable (V) domains of antibodies can be generated using standardmolecular biology and recombinant DNA techniques (see, e.g., Sambrook etal. (eds.), Molecular Cloning, A Laboratory Manual, 3^(rd) Edition, ColdSpring Harbor Laboratory Press, New York (2001)). Phage encoding avariable region with the desired specificity are selected for specificbinding to the desired antigen, and a complete or partial antibody isreconstituted comprising the selected variable domain. Nucleic acidsequences encoding the reconstituted antibody are introduced into asuitable cell line, such as a myeloma cell used for hybridomaproduction, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol.,235, 959-973 (1994).

The invention also provides antigen binding portions of any of theantibodies described herein. The antigen binding portion can be anyportion that has at least one antigen binding site, such as Fab,F(ab′)₂, dsFv, sFv, diabodies, and triabodies.

A single-chain variable region fragment (sFv) antibody fragment, whichconsists of a truncated Fab fragment comprising the variable (V) domainof an antibody heavy chain linked to a V domain of a light antibodychain via a synthetic peptide, can be generated using routinerecombinant DNA technology techniques (see, e.g., Janeway et al.,supra). Similarly, disulfide-stabilized variable region fragments (dsFv)can be prepared by recombinant DNA technology (see, e.g., Reiter et al.,Protein Engineering, 7, 697-704 (1994)). Antibody fragments of theinvention, however, are not limited to these exemplary types of antibodyfragments.

Also, the antibody, or antigen binding portion thereof, can be modifiedto comprise a detectable label, such as, for instance, a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase),and element particles (e.g., gold particles).

The inventive TCRs, polypeptides, proteins, (including functionalportions and functional variants thereof), nucleic acids, recombinantexpression vectors, host cells (including populations thereof), andantibodies (including antigen binding portions thereof), can be isolatedand/or purified. The term “isolated” as used herein means having beenremoved from its natural environment. The term “purified” as used hereinmeans having been increased in purity, wherein “purity” is a relativeterm, and not to be necessarily construed as absolute purity. Forexample, the purity can be at least about 50%, can be greater than 60%,70% or 80%, or can be 100%.

The inventive TCRs, polypeptides, proteins (including functionalportions and variants thereof), nucleic acids, recombinant expressionvectors, host cells (including populations thereof), and antibodies(including antigen binding portions thereof), all of which arecollectively referred to as “inventive TCR materials” hereinafter, canbe formulated into a composition, such as a pharmaceutical composition.In this regard, the invention provides a pharmaceutical compositioncomprising any of the TCRs, polypeptides, proteins, functional portions,functional variants, nucleic acids, expression vectors, host cells(including populations thereof), and antibodies (including antigenbinding portions thereof), and a pharmaceutically acceptable carrier.The inventive pharmaceutical compositions containing any of theinventive TCR materials can comprise more than one inventive TCRmaterial, e.g., a polypeptide and a nucleic acid, or two or moredifferent TCRs. Alternatively, the pharmaceutical composition cancomprise an inventive TCR material in combination with anotherpharmaceutically active agents or drugs, such as a chemotherapeuticagents, e.g., asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with theactive compound(s), and by the route of administration. Thepharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents, are well-known to thoseskilled in the art and are readily available to the public. It ispreferred that the pharmaceutically acceptable carrier be one which ischemically inert to the active agent(s) and one which has no detrimentalside effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive TCR material, as well as by the particular method used toadminister the inventive TCR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. The following formulations for parenteral, subcutaneous,intravenous, intramuscular, intraarterial, intrathecal, andinterperitoneal administration are exemplary and are in no way limiting.More than one route can be used to administer the inventive TCRmaterials, and in certain instances, a particular route can provide amore immediate and more effective response than another route.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inventive TCR material can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the inventive TCR material in solution.Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene glycol sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)). Preferably, when administering cells, e.g., dendritic cells,the cells are administered via injection.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the inventive TCRmaterials of the invention can be formulated as inclusion complexes,such as cyclodextrin inclusion complexes, or liposomes.

For purposes of the invention, the amount or dose of the inventive TCRmaterial administered should be sufficient to effect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to NY-ESO-1, or detect, treat orprevent cancer in a period of from about 2 hours or longer, e.g., 12 to24 or more hours, from the time of administration. In certainembodiments, the time period could be even longer. The dose will bedetermined by the efficacy of the particular inventive TCR material andthe condition of the animal (e.g., human), as well as the body weight ofthe animal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes of the invention, an assay, which comprises comparing theextent to which target cells are lysed or IFN-γ is secreted by T cellsexpressing the inventive TCR, polypeptide, or protein uponadministration of a given dose of such T cells to a mammal among a setof mammals of which is each given a different dose of the T cells, couldbe used to determine a starting dose to be administered to a mammal. Theextent to which target cells are lysed or IFN-γ is secreted uponadministration of a certain dose can be assayed by methods known in theart, including, for instance, the methods described herein as Example 3.

The dose of the inventive TCR material also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular inventive TCR material.Typically, the attending physician will decide the dosage of theinventive TCR material with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, inventive TCR material to beadministered, route of administration, and the severity of the conditionbeing treated. By way of example and not intending to limit theinvention, the dose of the inventive TCR material can be about 0.001 toabout 1000 mg/kg body weight of the subject being treated/day, fromabout 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1mg/kg body weight/day.

One of ordinary skill in the art will readily appreciate that theinventive TCR materials of the invention can be modified in any numberof ways, such that the therapeutic or prophylactic efficacy of theinventive TCR materials is increased through the modification. Forinstance, the inventive TCR materials can be conjugated either directlyor indirectly through a bridge to a targeting moiety. The practice ofconjugating compounds, e.g., inventive TCR materials, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J. DrugTargeting 3: 111 (1995) and U.S. Pat. No. 5,087,616. The term “targetingmoiety” as used herein, refers to any molecule or agent thatspecifically recognizes and binds to a cell-surface receptor, such thatthe targeting moiety directs the delivery of the inventive TCR materialsto a population of cells on which surface the receptor is expressed.Targeting moieties include, but are not limited to, antibodies, orfragments thereof, peptides, hormones, growth factors, cytokines, andany other natural or non-natural ligands, which bind to cell surfacereceptors (e.g., Epithelial Growth Factor Receptor (EGFR), T-cellreceptor (TCR), B-cell receptor (BCR), CD28, Platelet-derived GrowthFactor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).The term “bridge” as used herein, refers to any agent or molecule thatbridges the inventive TCR materials to the targeting moiety. One ofordinary skill in the art recognizes that sites on the inventive TCRmaterials, which are not necessary for the function of the inventive TCRmaterials, are ideal sites for attaching a bridge and/or a targetingmoiety, provided that the bridge and/or targeting moiety, once attachedto the inventive TCR materials, do(es) not interfere with the functionof the inventive TCR materials, i.e., the ability to bind to NY-ESO-1,or to detect, treat, or prevent cancer.

Alternatively, the inventive TCR materials can be modified into a depotform, such that the manner in which the inventive TCR materials isreleased into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of inventive TCR materials can be, forexample, an implantable composition comprising the inventive TCRmaterials and a porous or non-porous material, such as a polymer,wherein the inventive TCR materials is encapsulated by or diffusedthroughout the material and/or degradation of the non-porous material.The depot is then implanted into the desired location within the bodyand the inventive TCR materials are released from the implant at apredetermined rate.

It is contemplated that the inventive pharmaceutical compositions, TCRs(including functional portions or variants thereof), polypeptides,proteins, nucleic acids, recombinant expression vectors, host cells, orpopulations of cells can be used in methods of treating or preventingcancer. Without being bound to a particular theory or mechanism, theinventive TCRs are believed to bind specifically to NY-ESO-1, such thatthe TCR (or related inventive polypeptide or protein, or functionalportion or variant thereof) when expressed by a cell is able to mediatean immune response against the cell expressing NY-ESO-1. In this regard,the invention provides a method of treating or preventing cancer in amammal, comprising administering to the mammal any of the TCRs,polypeptides, or proteins described herein, any nucleic acid orrecombinant expression vector comprising a nucleotide sequence encodingany of the TCRs, polypeptides, proteins described herein, or any hostcell or population of cells comprising a recombinant vector whichencodes any of the TCRs, polypeptides, or proteins described herein, inan amount effective to treat or prevent cancer in the mammal.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

Also provided is a method of detecting the presence of cancer in amammal. The method comprises (i) contacting a sample comprising cells ofthe cancer any of the inventive TCRs, polypeptides, proteins, nucleicacids, recombinant expression vectors, host cells, populations of cells,or antibodies, or antigen binding portions thereof, described herein,thereby forming a complex, and detecting the complex, wherein detectionof the complex is indicative of the presence of cancer in the mammal.

With respect to the inventive method of detecting cancer in a mammal,the sample of cells of the cancer can be a sample comprising wholecells, lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detecting method, contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive TCRs, polypeptides,proteins, nucleic acids, recombinant expression vectors, host cells,populations of cells, or antibodies, or antigen binding portionsthereof, described herein, can be labeled with a detectable label suchas, for instance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer,cancer of the anus, anal canal, or anorectum, cancer of the eye, cancerof the intrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vulva, chronic lymphocyticleukemia, chronic myeloid cancer, colon cancer, esophageal cancer,cervical cancer, gastrointestinal carcinoid tumor. Hodgkin lymphoma,hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lungcancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynxcancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)),small intestine cancer, soft tissue cancer, stomach cancer, synovialcell sarcoma, testicular cancer, thyroid cancer, ureter cancer, andurinary bladder cancer. Preferably, the cancer is melanoma, breastcancer, lung cancer, prostate cancer, thyroid cancer, ovarian cancer, orsynovial cell sarcoma.

The mammal referred to in the inventive methods can be any mammal. Asused herein, the term “mammal” refers to any mammal, including, but notlimited to, mammals of the order Rodentia, such as mice and hamsters,and mammals of the order Logomorpha, such as rabbits. It is preferredthat the mammals are from the order Carnivora, including Felines (cats)and Canines (dogs). It is more preferred that the mammals are from theorder Artiodactyla, including Bovines (cows) and Swines (pigs) or of theorder Perssodactyla, including Equines (horses). It is most preferredthat the mammals are of the order Primates, Ceboids, or Simoids(monkeys) or of the order Anthropoids (humans and apes). An especiallypreferred mammal is the human.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Cell Lines

Melanoma lines 1300mel (NY-ESO-1⁺, HLA-A2⁺), 624.38mel (NY-ESO-1⁺,HLA-A2⁺), A375mel (NY-ESO-1⁺, HLA-A2⁺), 938mel (NY-ESO-1⁺, HLA-A2⁻),888mel (NY-ESO-1⁻, HLA-A2⁻), SK23mel (NY-ESO-1⁻, HLA-A2⁺), 1359mel(NY-ESO-1⁺, HLA-A2⁻), 1359-A2mel (NY-ESO-1⁺, HLA-A2⁺), 624mel(NY-ESO-1⁺, HLA-A2⁺), and 1390mel (NY-ESO-1⁺, HLA-A2⁺), were generatedfrom resected tumor lesions and were cultured in R10 medium consistingof RPMI 1640 supplemented with 10% fetal bovine serum, 2 mmol/LL-glutamine, 50 units/mL penicillin, and 50 μg/mL (Invitrogen) and 25mmol/L HEPES (GIBCO, Invitrogen). Other cell lines used included: thecervical cancer cell line Caski (NY-ESO-1⁺, HLA-A2⁺), (ATCC CRL-1550)the osteosarcoma cell line Saos2 (NY-ESO-1⁺, HLA-A2⁺), (ATCC HTB-85),and the neuroblastoma cell line SK NAS-A2 (NY-ESO-1⁺, HLA-A2⁺), (ATCCCRL-2137), the non-small cell lung cancer cell line H1299A2 (NY-ESO-1⁺,HLA-A2⁺), the breast carcinoma cell line MDA-MB-435S-A2 (NY-ESO-1⁺,HLA-A2⁺), (ATCC® HTB-129), all three of which were transduced withretroviral construct to express HLA-A*0201 (Navuaux et al., J. Virol.,70: 5701-05 (1996), Parkhurst et al., Clin. Cancer Res., 15: 169-180(2009), Robbins et al., J. Immunol., 180: 6116-31 (2008), Wargo et al.,Cancer Immunol. Immunother., 58: 394 (2009)), COS-A2-ESO, which wastransduced with a retroviral vector expressing the NY-ESO-1 gene, andCOS-A2-CEA, which was transduced with a retroviral vector expressing theCEA gene.

Example 1

This example demonstrates the identification of murine anti-NY-ESO-1 Tcell clones.

HLA-A2 transgenic mice were immunized with 100 μg of peptide(NY-ESO-1₁₅₇₋₁₆₅) and 120 μg of helper peptide (hepatitis B virus corepeptide (HBVc):128-140) in 100 μl Incomplete Freund's adjuvant (IFA)subcutaneously (s.c.) at the base of the tail (50 μg of NY-ESO-1₁₅₇₋₁₆₅peptide on each of two sides of the tail), followed by a boost one weeklater with the same immunization.

Day 0:

One week after the second immunization, splenocytes were harvested andstimulated in vitro with one of the following: (i) LPS-activated HLA-A2+splenocytes (3,000 rads) (“LPS blast”) pulsed with 1 μg/ml primingpeptide and 10 μg/ml human β2-microgobulin or (ii) T2 cells (17,000rads) pulsed with 1, 0.1 or 0.01 μg/ml peptide.

Day 7:

Bulk cultures were evaluated for specific reactivity via IFNγ secretionupon co-culture with one of the tumor cell lines set forth in Table 1.The results are shown in Table 1 (IFN-γ (pg/ml) post 1 bulk stimulation;“nt”=not tested). Because cytokine release was sometimes very high inresponse to T2 cells loaded with the HBV peptide, the underlined valuesfor tumor targets indicate twice the background values obtained withmedia alone and negative tumors, and the underlined values for peptidesindicate twice background values obtained with T2 and HBV peptide.

TABLE 1 LPS RNA copies blasts + 1 T2 + 1 T2 + 0.1 T2 + 0.01 per GAPDHμg/ml μg/ml μg/ml μg/ml HLA-A2 NY-ESO-1 (×100) peptide peptide peptidepeptide T2 + HBV + − nt 41 266  200 71 T2 + ESO: 157 + + nt 549  4505 4,464   406  media − − nt 22 53  69 41 888mel − − 0.02 36 133  110 88Sk23mel + − 0.01 24 76 134 17 1359mel − + 5.68 55 92  21 26 1359-A2 + +nt 22 98  73 48 A375mel + + 59.08  41 47 143 49 624mel + + 4.14 41 73200 22 1390mel + + nt — — — — 1363mel + + nt — — — — COS-A2-CEA + − nt32 94  67 56 COS-A2-ESO + + nt 33 92  65 61 293-A2-gp100 + − nt — — — —293-A2-ESO + + nt — — — —

Day 11:

Peptide/tumor reactive bulk cultures were cloned at 10 cells/well underthe following conditions (10 plates per condition): (i) irradiated T2cells (18,000 rads) pulsed with 1, 0.1, or 0.01 μg/ml peptide: 5×10⁴cells/well; (ii) irradiated C57BL/6 splenocyte feeders (3,000 rads):5×10⁴ cells/well; and (iii) 10 CU/ml IL-2.

Days 25-30:

Growth positive wells were selected and restimulated in 48-well platesunder the following conditions: (i) irradiated T2 cells (18,000 rads)pulsed with 1, 0.1, or 0.01 μg/ml peptide: 2×10⁵ cells/well; (ii)irradiated C57BL/6 splenocyte feeders (3,000 rads): 1×10⁶ cells/well;and (iii) 10 CU/ml IL-2.

Days 37-44:

Clones were evaluated for specific reactivity via IFNγ secretion uponco-culture with the tumor cell lines set forth in Table 2. Tumor cellswere treated with IFNγ (20 ng/ml) and tumor necrosis factor alpha (3ng/ml) overnight prior to the assay.

The splenocytes stimulated with LPS-activated HLA-A2+ splenocytes (3,000rads) pulsed with 1 μg/ml priming peptide and 10 μg/ml humanβ2-microgobulin on Day 0 produced 8 out of 960 growth positive wells.Data for the two most reactive clones are shown in Table 2 (post 1 bulkstimulation; IFN-γ (pg/ml)).

TABLE 2 RNA copies HLA- NY- per GAPDH A2 ESO-1 (×100) B H T2 + + − nt393 283 HBV T2 + + + nt >14,000    >14,000    ESO: 157 media − − nt 404292 888mel − − 0.02 386 288 Sk23mel + − 0.01 — — 1359mel − + 5.68 354285 1359-A2 + + nt 11,781   16,436   A375mel + + 59.08  383 1,954  624mel + + 4.14 363 14,298   1390mel + + nt 288 17,567   1363mel + + nt3,582   — COS-A2- + − nt 348 289 CEA COS-A2- + + nt >14,000   >14,000    ESO 293-A2- + − nt 373 274 gp100 293-A2- + + nt 335 8,813  ESO

Days 46-49:

Clones of interest were restimulated in 24-well plates under thefollowing conditions: (i) irradiated T2 cells (18,000 rads) pulsed with1, 0.1, or 0.01 μg/ml peptide: 5×10⁵ cells/well; (ii) irradiated C57BL/6splenocyte feeders (3,000 rads): 1×10⁶ cells/well; and (iii) 10 CU/mlIL-2. Restimulated clones were flash-frozen for RNA preparation.

Example 2

This example demonstrates the identification of murine anti-NY-ESO-1 Tcell clones.

HLA-A2 transgenic mice were immunized and the splenocytes wereharvested, stimulated, and evaluated for specific reactivity asdescribed in Example 1.

Day 11:

The bulk cultures were restimulated in 24-well plates under thefollowing conditions: (i) irradiated T2 cells (18,000 rads) pulsed with1, 0.1, or 0.01 μg/ml peptide: 4×10⁵ cells/well; (ii) irradiated C57BL/6splenocyte feeders (3,000 rads): 1×10⁶ cells/well; and (iii) 10 CU/mlIL-2.

Day 19:

The bulk cultures (post two stimulations) were evaluated for specificreactivity via IFN-γ secretion upon co-culture with the tumor cell linesset forth in Table 3. Tumor cells were treated with IFNγ (20 ng/ml) andtumor necrosis factor alpha (3 ng/ml) overnight prior to the assay. Theresults are shown in Table 3 (IFN-γ (pg/ml)).

TABLE 3 Post 2 bulk stims. LPS RNA copies blasts + 1 T2 + 1 T2 + 0.1T2 + 0.01 per GAPDH μg/ml μg/ml μg/ml μg/ml HLA-A2 NY-ESO-1 (×100)peptide peptide peptide peptide T2 + HBV + − nt 195 2,860   16,156  1,058   T2 + ESO: 157 + + nt 79,524   72,730   47,871   1,899   media −− nt 137 131 156 406 888mel − − 0.02  40 201 112 562 Sk23mel + − 0.01 79 245 562 424 1359mel − + 5.68  73 169 188 357 1359-A2 + + nt 966 3201,597   258 A375mel + + 59.08  150 176 258 332 624mel + + 4.14 320 144697 301 1390mel + + nt — — — — 1363mel + + nt — — — — COS-A2-CEA + − nt226 369 400 308 COS-A2-ESO + + nt 424 351 326 295 293-A2-gp100 + − nt —— — — 293-A2-ESO + + nt — — — —

Day 21:

Bulk cultures were restimulated in 24-well plates under the followingconditions: (i) irradiated T2 cells (18,000 rads) pulsed with 1, 0.1, or0.01 μg/ml peptide: 5×10⁵ cells/well; (ii) irradiated C57BL/6 splenocytefeeders (3,000 rads): 1×10⁶ cells/well; and (iii) 10 CU/ml IL-2.

Day 30:

The bulk cultures (post three stimulations) were evaluated for specificreactivity via IFN-γ secretion upon co-culture with the tumor cell linesset forth in Table 4. Tumor cells were treated with IFNγ, (20 ng/ml) andtumor necrosis factor alpha (3 ng/ml) overnight prior to the assay. Theresults are shown in Table 4 (IFN-γ (pg/ml); * indicates bulk culturesthat were cloned after three bulk stimulations).

TABLE 4 * LPS TE8 RNA copies blasts + 1 * T2 + 1 T2 + 0.1 T2 + 0.01(human per GAPDH μg/ml μg/ml μg/ml μg/ml T cell HLA-A2 NY-ESO-1 (×100)peptide peptide peptide peptide clone) T2 + HBV + − nt 1,794 4,700  28,797  23,897   16 T2 + ESO: 157 + + nt 78,316  63,793   96,164  19,69816,254  media − − nt 1,992 389 8,856 10,711    7 888mel − − 0.02 1,268188 6,611 7,614   13 Sk23mel + − 0.01   662 202 7,585 6,225   69 1359mel− + 5.68   623  64 5,684 7,026   996 1359-A2 + + nt 27,572  7,774  10,324  7,204 7,936 A375mel + + 59.08  1,263 342 7,232 9,425 4,095624mel + + 4.14 14,098  3,211   10,061  8,727 3,262 1390mel + + nt   852179 5,966 6,191 7,123 1363mel + + nt 42,970  15,673   20,398  9,95812,149  COS-A2-CEA + − nt   981 119 3,995 6,744   18 COS-A2-ESO + + nt19,523  3,334   9,116 8,187 14,662  293-A2-gp100 + − nt — — — — —293-A2-ESO + + nt — — — — —

Day 33:

Selected peptide/tumor reactive bulk cultures (post three stimulations)were cloned at 10 cells/well as described for Day 11 of Example 1.

Days 45-48:

Growth positive wells were screened for peptide reactivity via IFN-γsecretion upon co-culture with the tumor cell lines set forth in Table5. Tumor cells were treated with INFγ (20 ng/ml) and tumor necrosisfactor alpha (3 ng/ml) overnight prior to the assay.

The splenocytes stimulated with LPS-activated HLA-A2+ splenocytes (3,000rads) pulsed with 1 μg/ml priming peptide and 10 μg/ml humanβ2-microgobulin on Day 0 produced 33 out of 960 growth positive wells.Data for the four most reactive clones (Nos. 2, 5, 6, and 8) are shownin Table 5 (post 3 bulk stimulations; IFN-γ (pg/ml)). The splenocytesstimulated with T2 cells (17,000 rads) pulsed with 1 μg/ml peptideproduced 104 out of 960 growth positive wells. Data for the four mostreactive clones (Nos. 1, 50, 51, and 63) are shown in Table 5.

TABLE 5 RNA copies per GAPDH HLA-A2 NY-ESO-1 (×100) 2 5 6 8 T2 + HBV + −nt 235 317 223 344 T2 + ESO: 157 + + nt >14,000    >14,000    >14,000   >14,000    media − − nt 221 314 231 563 888mel − − 0.02 210 316 208 288Sk23mel + − 0.01 216 309 210 297 1359mel − + 5.68 243 383 206 4221359-A2 + + nt 1,981   6,027   2,523   1,845   A375mel + + 59.08  523388 232 1,455   624mel + + 4.14 7,879   1,931   2,527   8,595  1390mel + + nt 1,124   17,567   352 900 1363mel + + nt — — — —COS-A2-CEA + − nt — — — — COS-A2-ESO + + nt — — — — 293-A2-gp100 + − nt— — — — 293-A2-ESO + + nt — — — — 1 50 51 63 T2 + HBV 306 335 270 239T2 + ESO: 157 >14,000    >14,000    >14,000    >14,000    media 266 232258 222 888mel 234 241 244 208 Sk23mel 278 208 222 207 1359mel 347 271212 229 1359-A2 1,477   3,661   1,965   2,272   A375mel 556 557 556 737624mel 4,629   4,487   2,978   7,139   1390mel 517 379 914 716 1363mel —— — — COS-A2-CEA — — — — COS-A2-ESO — — — — 293-A2-gp100 — — — —293-A2-ESO — — — —

Days 46-49:

Peptide reactive clones were restimulated in 24-well plates as describedfor Day 21 of this example. Restimulated clones were flash-frozen forRNA preparation.

Example 3

This example demonstrates the isolation of a murine anti-NY-ESO-1 TCRand the specific reactivity of the isolated TCR against NY-ESO-1.

The TCR from five clones, (namely, clones B, H, 5, 6, 1, 50, and 63)were isolated. The nucleotide sequence (RNA) encoding the TCR of eachclone was isolated, sequenced, and transfected into human peripheralblood mononuclear cells (PBMC) from Patients 1 and 2. The transfectedcells were stimulated with OKT3 and IL-2 and cultured alone (media) orco-cultured with T2 cells pulsed with control (HBV) peptide, T2 cellspulsed with NY-ESO-1₁₅₇₋₁₆₅ peptide, COA-A2-CEA (NY-ESO-1⁻), COS-A2-ESO(NY-ESO-1⁺), or one of various melanoma tumor cell lines 888mel(NY-ESO-1⁻), Sk23mel (NY-ESO-1⁻), A375mel (NY-ESO-1⁺), 1363mel(NY-ESO-1⁺), 1390 (NY-ESO-1⁺), or 624 (NY-ESO-1⁺). IFNγ secretion wasmeasured. The results are shown in Table 6 (IFNγ (pg/ml)).

T2 + ESO: COS-A2- COS-A2- T2 + HBV 157 media 888 Sk23 1363 1390 A375 624CEA ESO HLA-A2 − − − − + + + + + + + NY-ESO-1 − + − − − + + + + − +Patient 1 GFP 326    180 8 198 134 220 325 632 32 94 74 Avidex TCR235 >10,000 7 188 81 >10,000    1637  5752  531  52 2320 TRAV7D-4/TRBV19¹ 664 >10,000 7 185 102 2262  489 893 39 72 449 TRAV13D-2/TRBV14² 197 >10,000 8 152 88 134 159 338 26 75 55TRAV7D-3/TRBV14³ 155    366 7 129 112 122 171 378 28 68 71TRAV6D/TRBV26⁴ 198 >10,000 11 255 156 >10,000    3859  9973  782  913872  TRAV7D-3/TRBV26⁵ 190   1269 0 208 107 246 189 509 34 79 104 Patient 2 GFP 26    30 2 47 27  22  62  98  8 16 19 Avidex TCR50 >10,000 4 32 22 2484  208 192 150  11 588  TRAV7D-4/TRBV19¹183 >10,000 2 34 15 149  47  39  7 13 90 TRAV13D-2/TRBV14² 22   7898 927 13  20  42  58 11 22 17 TRAV7D-3/TRBV14³ 24    23 11 28 13  21  30 39  5 7 13 TRAV6D/TRBV26⁴ 63 >10,000 39 77 40 3597  777 344 133  32683  TRAV7D-3/TRBV26⁵ 10    77 0 28 17  27  33  51  5 16 40¹TRAV7D-4/TRBV19: Clone ESO (1 stim LPS) B (Table 2 above)²TRAV13D-2/TRBV14: Clone ESO (1 stim LPS) H (Table 2 above)³TRAV7D-3/TRBV14: Clone ESO (3 stim LPS) 5 (Table 5 above)⁴TRAV6D/TRBV26: Clones ESO (3 stim LPS) 6; ESO (3 stim T2) 1; ESO (3stim T2) 63 (Table 5 above) ⁵TRAV7D-3/TRBV26: Clone ESO (3 stim T2) 50(Table 5 above)

As shown in Table 6, the TRAV6D/TRBV26 (SEQ ID NOs: 11 and 12) TCRprovided the highest specific anti-NY-ESO-1 reactivity and was chosenfor further study.

The nucleotide sequence (RNA) encoding the TRAV6D/TRBV26 (SEQ ID NOs: 11and 12) TCR was transfected into human PBMC from Patients 3 and 4. Thetransfected cells were positively selected for CD8+ and CD4+ cells,stimulated with OKT3 and IL-2, and cultured alone (media) or co-culturedwith T2 cells pulsed with control (HBV) peptide, T2 cells pulsed withvarious concentrations of NY-ESO-1₁₅₇₋₁₆₅ peptide, COS-A2-ESO(NY-ESO-1⁺), COA-A2-CEA (NY-ESO-1⁻), or one of various melanoma tumorcell lines 888mel (NY-ESO-1⁻), Sk23mel (NY-ESO-1⁻), A375mel (NY-ESO-1⁺),1363mel (NY-ESO-1⁺), or 624 (NY-ESO-1⁺). IFNγ secretion was measured andthe results are shown in Table 7 (INFγ (pg/ml)).

TABLE 7 T2 + HBV ---------T2 + ESO:157-165------------------------------------- 888 Sk23 10⁻⁶ 10⁻¹² 10⁻¹¹10⁻¹⁰ 10⁻⁹ 10⁻⁸ 10⁻⁷ 10⁻⁶ A2− A2+ g/ml g/ml g/ml g/ml g/ml g/ml g/mlg/ml media ESO− ESO− Patient 3 CD8+ TRAV6D/ 243 238 1075   4257 6170 8996 5087 6142 122 90 70 TRBV26 GFP 86 47 27   29   9   17  18  25 1219 5 CD4+ TRAV6D/ 127 67 69  1836 12835  19161 12495  14641  173 56 43TRBV26 Avidex 6 0 1  40 1147  2455 3538 5783 17 0 0 GFP 10 0 0   0   0  0   0   0 10 5 0 Patient 4 CD8+ TRAV6D/ 15 55 931  1713 2670  37212988 2878 0 0 0 TRBV26 GFP 42 0 0   0   0   0   5   0 0 0 6 CD4+ TRAV6D/38 1 58  5774 18051  19691 >20000  >20000  21 7 0 TRBV26 Avidex 25 0 0 200 2923 15574 >20000  18293  8 0 0 GFP 12 0 0   0   0   0  10  30 0 00 COS-A2 COS-A2 A375 624 1363 CEA ESO A2+ A2+ A2+ A2+ A2+ ESO+ ESO+ ESO+ESO− ESO+ Patient 3 CD8+ TRAV6D/ 419  125 1194  56 1041  TRBV26 GFP 18 127 11 20 CD4+ TRAV6D/ 250  84 361  30 179  TRBV26 Avidex 25 9 26 10 14GFP 43 18 47 4  7 Patient 4 CD8+ TRAV6D/ 48 15 262  0 380  TRBV26 GFP  50  0 2  0 CD4+ TRAV6D/ 109  36 476  2 241  TRBV26 Avidex 37 0 60 0 23GFP 28 14 42 0  0

As shown in Table 7, the cells transfected with the TRAV6D/TRBV26 (SEQID NOs: 11 and 12) TCR specifically recognized NY-ESO-1+ melanoma tumorcells, as measured by IFNγ secretion.

Example 4

This example demonstrates the reactivity of human CD8+ and CD4+ T cellstransfected with a murine anti-NY-ESO-1 TCR upon co-culture withdendritic cells pulsed with NY-ESO-1 peptide.

CD8+ (FIG. 1A) or CD4+ (FIG. 1B) human T cells were transfected with amurine anti-NY-ESO-1 TCR (TRAV6D/TRBV26 (SEQ ID NOs: 11 and 12)) or ahuman anti-NY-ESO-1 TCR. The transfected cells were co-cultured withdendritic cells pulsed with various concentrations of NY-ESO-1₁₅₇₋₁₆₅peptide, and INFγ secretion was measured.

As shown in FIGS. 1A and 1B, CD8+ and CD4+ human T cells transfectedwith a murine anti-NY-ESO-1 TCR (TRAV6D/TRBV26 (SEQ ID NOs: 11 and 12))were reactive against dendritic cells pulsed with NY-ESO-1₁₅₇₋₁₆₅peptide, as measured by IFNγ secretion. CD8+ human T cells transfectedwith a murine anti-NY-ESO-1 TCR (TRAV6D/TRBV26 (SEQ ID NOs: 11 and 12))were more reactive against dendritic cells pulsed with NY-ESO-1₁₅₇₋₁₆₅peptide, as measured by IFNγ secretion, as compared to CD8+ cellstransfected with a human anti-NY-ESO-1 TCR.

Example 5

This example demonstrates the reactivity of human CD8+ and CD4+ T cellstransfected with a murine anti-NY-ESO-1 TCR upon co-culture withmelanoma tumor cells.

CD8+ (FIG. 2A) or CD4+ (FIG. 2B) human T cells were transfected with amurine anti-NY-ESO-1 TCR (TRAV6D/TRBV26 (SEQ ID NOs: 11 and 12)) or ahuman anti-NY-ESO-1 TCR. The transfected cells were cultured alone(media) or co-cultured with T2 cells pulsed with control peptide, T2cells pulsed with NY-ESO-1₁₅₇₋₁₆₅ peptide, COA-A2-CEA (NY-ESO-1⁻),COS-A2-ESO (NY-ESO-1⁺), or one of various melanoma tumor cell lines888mel (NY-ESO-1⁻), Sk23mel (NY-ESO-1⁻), A375mel (NY-ESO-1⁺), or 1363mel(NY-ESO-1⁺). IFNγ secretion was measured.

As shown in FIGS. 2A and 2B, CD8+ and CD4+ human T cells transfectedwith a murine anti-NY-ESO-1 TCR (TRAV6D/TRBV26 (SEQ ID NOs: 11 and 12))specifically recognized NY-ESO-1+ melanoma tumor cells, as measured byIFNγ secretion. CD8+ and CD4+ human T cells transfected with a murineanti-NY-ESO-1 TCR (TRAV6D/TRBV26 (SEQ ID NOs: 11 and 12)) were morereactive against NY-ESO-1+ tumor cell lines, as measured by IFNγsecretion, as compared to CD8+ and CD4+ cells transfected with a humananti-NY-ESO-1 TCR.

Example 6

This example demonstrates the reactivity of human CD8+ and CD4+ T cellstransfected with a wild-type or codon-optimized nucleotide sequenceencoding a murine anti-NY-ESO-1 TCR upon co-culture with melanoma tumorcells.

A wild-type (SEQ ID NOs: 19 and 20) or codon-optimized (SEQ ID NOs: 15and 16) nucleotide sequence (RNA) encoding the murine anti-NY-ESO-1 TCR(TRAV6D/TRBV26) was transfected into CD8+ or CD4+ human PBMC fromPatients 5 and 6. The transfected cells were positively selected forCD8+ and CD4+ cells, stimulated with OKT3 and IL-2, and cultured alone(media) or co-cultured with T2 cells pulsed with control (HBVc) peptide,T2 cells pulsed with various concentrations of NY-ESO-1₁₅₇₋₁₆₅ peptide,COA-A2-CEA (NY-ESO-1⁻), COS-A2-ESO (NY-ESO-1), or one of variousmelanoma tumor cell lines 888mel (NY-ESO-1⁻), Sk23mel (NY-ESO-1⁺),A375mel (NY-ESO-1⁺), 1363mel (NY-ESO-1⁻), A375 (NY-ESO-1⁻), or 624mel(NY-ESO-1⁺). IFNγ secretion was measured. The results are shown in Table8 (IFNγ (pg/ml)).

TABLE 8 ----------Patient 5 CD4---------- ---------Patient 5CD8------------ -------Patient 6 CD4----------- Codon wild Codon wildCodon wild optimized type GFP optimized type GFP optimized type GFPmedia 103  62 6 43  60 1  0  0 0 T2 + HBVc 58 37 4 71 108 82  2 17 7T2 + 10⁻¹² M ESO 44 32 0 67  86 46 13 15 6 T2 + 10⁻¹¹ M ESO 83 42 0 52150 30 15 22 7 T2 + 10⁻¹⁰ M ESO 69 27 3 221  175 43 13 18 4 T2 + 10⁻⁹ MESO 232  60 0 3149  2465  20 728  555  6 T2 + 10⁻⁸ M ESO 5158  2979  013308   13394  56 15468   12906   7 T2 + 10⁻⁷ M ESO 13987   9362  1>20000   >20000   37 23981   21321   8 T2 + 10⁻⁶ M ESO 15345   9417  0>20000   >20000   51 26802   25225   8 888mel (A2− ESO−) 54 30 8 55  9837 22  9 8 Sk23mel (A2+ 30 34 7  8  26 29  3  0 3 ESO−) A375 (A2+ ESO+)239  204  75 525  925 55 114  146  82 624mel (A2+ ESO+) 102  81 57 176 211 24 44 42 6 1363mel (A2+ ESO+) 605  443  176 964  1421  28 525  576 153 COS-A2-CEA 68 66 22 51  83 39  8 17 16 COS-A2-ESO 429  92 2 1322 1436  22 1213  431  17 -------Patient 6 CD8------------- Codon wildoptimized type GFP media media  0  0 0 3 T2 + HBVc  3 12 9 8 T2 + 10⁻¹²M ESO  6  9 14 0 T2 + 10⁻¹¹ M ESO  20 23 2 0 T2 + 10⁻¹⁰ M ESO 193 176  00 T2 + 10⁻⁹ M ESO 1979  3066  4 0 T2 + 10⁻⁸ M ESO 5382  11217   0 0 T2 +10⁻⁷ M ESO 8914  13127   0 0 T2 + 10⁻⁶ M ESO 14734  19766   2 0 888mel(A2− ESO−)  36 58 37 17 Sk23mel (A2+  8 29 11 0 ESO−) A375 (A2+ ESO+)190 480  22 0 624mel (A2+ ESO+)  89 125  0 0 1363mel (A2+ ESO+) 4261079  20 nt COS-A2-CEA  4 13 9 4 COS-A2-ESO 872 1372  10 0

As shown in Table 8, CD8+ and CD4+ human T cells transfected with awild-type or codon-optimized nucleotide sequence encoding a murineanti-NY-ESO-1 TCR (TRAV6D/TRBV26) specifically recognized NY-ESO-1+melanoma tumor cells, as measured by IFNγ secretion.

Example 7

This example demonstrates the reactivity of human CD8+ T cellstransfected with a wild-type nucleotide sequence encoding a murineanti-NY-ESO-1 TCR upon co-culture with melanoma and non-melanoma tumorcells.

A nucleotide sequence (RNA) (SEQ ID NO: 19 and 20) encoding the murineanti-NY-ESO-1 TCR (TRAV6D/TRBV26) was electroporated into CD8+ human Tcells from Patients 7 and 8. Untransfected cells (mock) or transfectedcells were positively selected for CD8+ T cells, stimulated with OKT3and IL-2, and cultured alone (media) or co-cultured with T2 cells pulsedwith control (HBVc) peptide; T2 cells pulsed with various concentrationsof NY-ESO-1₁₅₇₋₁₆₅ peptide; COA-A2-CEA (NY-ESO-1⁻); COS-A2-ESO(NY-ESO-1⁺); one of various melanoma tumor cell lines 888mel(NY-ESO-1⁻), Sk23mel (NY-ESO-1⁻), A375mel (NY-ESO-1⁺), 1363mel(NY-ESO-1⁺), A375 (NY-ESO-1⁺); osteogenic sarcoma cell line Saos2(NY-ESO-1⁺); glioma cell line LN-18 (NY-ESO-1⁺); Ewing's sarcoma cellline TC-71 (NY-ESO-1⁺); neuroblastoma cell lines SKN AS (NY-ESO-1⁺) orSKN AS-A2 (NY-ESO-1⁺); or breast cancer cell lines MDA 453S (NY-ESO-1⁺)or MDA 453S-A2 (NY-ESO-1⁺). IFNγ secretion was measured. The results areshown in Table 9 (IFNγ (pg/ml)).

TABLE 9 --Patient 7 CD8--- --Patient 8 CD8--- ESO a/b mock ESO a/b mockmedia media  21 0  0 6 0 T2 + HBVc  64 60  88 50 0 T2 + 10−12 M ESO  5058 107 59 0 T2 + 10−11 M ESO  66 51 209 71 0 T2 + 10−10 M ESO 332 661704  50 0 T2 + 10−9 M ESO 3142  51 10886  55 0 T2 + 10−8 M ESO 6505  52>20000   58 0 T2 + 10−7 M ESO 6764  42 >20000   51 0 T2 + 10−6 M ESO6550  55 >20000   58 0 888mel (A2− ESO −) melanoma  59 45 142 133 0Sk23mel (A2+ ESO−) melanoma  79 54  31 59 0 A375mel (A2+ ESO+) melanoma2986  240 1984  93 0 1363mel (A2+ ESO+) melanoma 1889  119 9858  137 0Saos2 (A2+ ESO+) osteogenic sarcoma 248 34 1253  28 0 LN-18 (A2+ ESO+)glioma 123 21 224 34 0 TC-71 (A2+ ESO+) Ewing's sarcoma 159 116 183 1273 SKN AS (A2− ESO+) neuroblastoma 542 328 199 207 2 SKN AS - A2 (A2+ESO+) neuroblastoma 148 38 1004  39 0 MDA 453S (A2− ESO+) breast cancer448 311 177 230 0 MDA 453S -A2 (A2+ ESO+) breast cancer 111 50 610 39 0COS-A2-CEA (A2+ ESO−)  45 51  50 63 7 COS-A2-ESO (A2+ ESO+) 588 34 4109 63 0

As shown in Table 9, CD8+ human T cells transfected with a nucleotidesequence encoding a murine anti-NY-ESO-1 TCR (TRAV6D/TRBV26)specifically recognized NY-ESO-1+ melanoma, osteogenic sarcoma, Ewing'ssarcoma, neuroblastoma, and breast cancer tumor cells, as measured byIFNγ secretion.

Example 8

This example demonstrates the preparation of recombinant expressionvectors encoding a murine anti-NY-ESO-1 TCR.

A retroviral vector comprising DNA encoding wild-type humananti-NY-ESO-1 TCR (1G4), 1G4 TCR having a double substitution within theCDR3α chain in which leucine and tyrosine are substituted for threonineat position 95 (1G4-LY) (Robbins et al., J. Clin. Oncol., 29: 917-924(2011); Robbins et al., J. Immunol., 180: 6116-6131 (2008)), or murineanti-NY-ESO-1 TCR (TRAV6D/TRBV26) (SEQ ID NOs: 11 and 12) were clonedinto a MSGVI retroviral backbone and transformed into TOP10 cells. Apicornavirus 2A peptide (SEQ ID NO: 13) linked the alpha and betachains. Two vectors encoding the murine TRAV6D/TRBV26 TCR were made: onecontained the nucleotide sequence encoding the alpha chain located 5′ ofthe nucleotide sequence encoding the beta chain (mESOαβ) (SEQ ID NO:17), and one contained the nucleotide sequence encoding the beta chainlocated 5′ of the nucleotide sequence encoding the alpha chain (mESOβα)(SEQ ID NO: 18). The presence of the inserts encoding the alpha and betachains of the TCR was confirmed by digestion with Nco I and Not Irestriction enzymes. DNA was generated from one clone for each of thehuman and murine TCRs by maxiprep.

DNA from the 1G4 TCR and 1G4-LY vectors was transfected into 293GP cellsto collect supernatant and transduce PBL in subsequent transductionexperiments. A vector encoding GFP was used as a control.

Example 9

This example demonstrates the transduction efficiency of a murineanti-NY-ESO-1 TCR.

Peripheral blood lymphocytes (PBL) were stimulated with OKT3 on Day 0(S1). The PBL were transduced with the 1G4, 1G4-LY, mESOαβ, or mESOβαTCR vector of Example 8 on Days 3 and 4. On Days 7-11, transductionefficiency was evaluated by fluorescence-activated cell sorting (FACS).An antibody recognizing the variable region of the murine TCR (VB13.1)and an antibody recognizing the constant region of the murine TCR (mB)were used for the FACS. The FACS was performed 7 to 11 days after firststimulation (S1D7-S1D11). The results are summarized in Table 10 below.

TABLE 10 % VB13.1, mB+ cells pre-rapid expansion (REP) (for 5 donors)(S1D7-S1D11) Untransduced (UT) 0-6 Green fluorescent protein (GFP) 67-90.5 1G4 TCR 62-85 1G4-LY TCR 37-85 mESOαβ TCR 56-90 mESOβα TCR56-91

As shown in Table 10, PBL transduced with the mESOαβ or mESOβα TCRvector were transduced with similar efficiency as compared to thevectors encoding the 1G4 and 1G4-LY TCRs.

Example 10

This example demonstrates the reactivity of cells transduced with avector encoding a murine anti-NY-ESO-1 TCR.

PBL from five donors were stimulated and either untransduced ortransduced with vectors encoding GFP or the 1G4-LY, mESOαβ, or mESOβαTCR as described in Example 9. Transduced PBL were co-cultured with oneof the various tumor cell lines listed in Table 11A or 11B below or withT2 cells pulsed with SSX peptide, no peptide (T2), or one of the variousconcentrations of NY-ESO-1₁₅₇₋₁₆₅ peptide listed in Table 12 below. IFNγsecretion was measured by enzyme-linked immunosorbent assay (ELISA) of24 hour supernatant from co-cultures. ELISA was performed 6, 7, or 10days following first stimulation (S1D6, S1D7, and S1D10). The resultsare shown in Tables 11A, 11B, and 12 (IFNγ pg/ml). Transduction (Td)efficiency was based on FACS analysis of Vβ13.1+mβ+ cells.

TABLE 11A % td COS-A2- H1299- COAS-A2- efficiency 888 938 gp100 624.38A2 A375 ESO 1300 Patient 1 (Dilution 1:10; S1D7) Untransduced (UT) N/A12 0 99  22  17 132  24 61 GFP 90  0 0 91  17  65  95  0 33 1G4-LY TCR85 13 0 99 3316  8592 3059  1391  5732  mESOαβ TCR 83 239  284  156 5523  9616 4430  1872  6922  mESOβα TCR 83 329  326  42 6721  9898 4178 2270  7798  Patient 2 (Dilution 1:5; S1D7) Untransduced (UT) N/A 44 43 46  44  42  53  46 42 GFP 67 42 42  43  44  40  50  41 41 1G4-LY TCR 6353 43  44 346 1446 451 132 352  mESOαβ TCR 56 43 44  45 372 1259 415 108323  mESOβα TCR 56 43 46  45 327 1236 412 136 625  Patient 3 (Dilution1:5; S1D10) Untransduced (UT) N/A  0 0  0  0   0  48  39 — GFP 63  0 0 0  0   0  0  0 — 1G4-LY TCR 76  0 305   0 546  770 1020  332 — mESOαβTCR 63  0 0  0 111  38 292  13 — mESOβα TCR 60  0 0  0 413  540 819 283—

TABLE 11B % Td COS-A2 COS-A2- efficiency Media 888 938 gp100 624.38H1299-A2 A375 ESO Patient 4 (Dilution 1:5; S1D6) Untransduced NA 52 15535 294 195 122 476 212 (UT) GFP 91% 34 91 22 78 106 62 306 50 1G4-LY TCR75% 86 113 54 313 2172 7187 1671 2394 mESOαβ TCR 84% 41 131 98 185 19465744 1064 1490 mESOβα TCR 85% 52 130 110 164 2812 7163 1262 1676 Patient5 (Dilution 1:5; S1D6) Untransduced NA 42 23 12 88 9 8 107 64 (UT) GFP87% 15 20 15 76 0 11 98 62 1G4-LY TCR 25% 63 16 10 20 1172 3156 918 430mESOαβ TCR 74% 19 55 22 57 444 917 233 372 mESOβα TCR 74% 15 32 20 24810 2417 603 380

TABLE 12 % td efficiency SSX T2 1 μg/ml 100 ng/ml 10 ng/ml 1 ng/ml 0.1ng/ml Patient 1 (Dilution 1:5; S1D7) Untransduced (UT) NA 0 0   0   0  0  0  0 GFP 90 0 0   0   0   0  0  0 1G4 TCR 85 0 0 2452 2475 1525 85199 1G4-LY TCR 85 0 0 2173 1599 1087 677  0 mESOαβ TCR 83 0 0 2825 22101904 1060  278  mESOβα TCR 83 0 0 3018 2403 2020 1212  522  Patient 2(Dilution 1:5; S1D7) Untransduced (UT) NA 22 20  52  24  21  33 53 GFP67 17 38  33  39  31  21 20 1G4 TCR 62 19 15 1,963  1,104   860 397 411G4-LY TCR 63 15 19 2441 1280  879 291 40 mESOαβ TCR 56 20 42 4645 18541529 535 92 mESOβα TCR 56 38 27 7091 2302 1336 348 262 

Cells transduced with the mESOαβ or the mESOβα TCR vectors specificallyrecognized NY-ESO-1⁺/HLA-A*0201⁺ target tumor cell lines but notHLA-A*0201⁻/NY-ESO-1⁺ or HLA-A*0201⁺/NY-ESO-1⁻ cell lines as measured byIFNγ secretion (Tables 11A and 11B). Cells transduced with the mESOαβ orthe mESOβα TCR vectors specifically recognized T2 cells pulsed withNY-ESO-1 peptide as measured by IFNγ secretion (Table 12). The NY-ESO-1specific recognition was consistent among cells from five differentdonors. Functionality of the cells transduced with the murineanti-NY-ESO-1 TCR was comparable to that of cells transduced with humananti-NY-ESO-1 TCR. Functionality of the cells transduced with the mESOβαTCR vector was slightly higher as compared to that of the cellstransduced with the mESOαβ TCR vector. PBL transduced with either mESOαβor the mESOβα TCR vectors recognized T2 cells pulsed with as little as 1ng/mL, indicating that both mTCRs are relatively high avidity receptors.Co-culture of PBL expressing mESOαβ or the mESOβα TCR vectors withcontrol T2 cells that were not pulsed with any peptide producedbackground levels of IFN-γ. The cells of Patient 1 transduced withmESOβα TCR had higher levels of IFN-γ secretion when compared to thecells transduced with mESOαβ TCR for the same level of peptide. Thecells of Patient 2 transduced with mESOβα TCR had higher levels of IFN-γsecretion when compared to the cells transduced with mESOαβ TCR for thesame level of peptide for peptide concentrations 1 μg/ml, 100 ng/ml, and0.1 ng/ml.

Example 11

This example demonstrates that cells transduced with a vector encoding amurine anti-NY-ESO-1 TCR maintain expression of the murine anti-NY-ESO-1TCR following expansion of the numbers of cells.

PBL from two donors were stimulated and either untransduced ortransduced with vectors encoding GFP or the 1G4, 1G4-LY, mESOαβ, ormESOβα TCR as described in Example 9. The numbers of PBL were expandedas described in Riddell et al., Science, 257:238-241 (1992) and Dudleyet al., Cancer J. Sci. Am., 6:69-77 (2000). Generally, the numbers ofPBL were expanded up to 3 logs using soluble OKT3, irradiated feedercells, and high-dose IL-2. Expression of murine anti-NY-ESO-1 TCR byexpanded numbers (expanded once) of cells was measured by FACS twice (onDays 10 and 20). The results are summarized in Table 13 (% VB13.1, mB+cells following expansion).

TABLE 13 Donor 1 2 D10 D20 D10 D20 UT 0 <1 0 <1 GFP 88 87 42 70 1G4 TCR59 80 50 59 1G4-LY TCR 76 88 37 60 mESOαβ TCR 82 76 62 46 mESOβα TCR 8274 62 50

As shown in Table 13, PBL transduced with the mESOαβ or mESOβα TCRvector maintained expression of the murine anti-NY-ESO-1 TCR followingexpansion of the numbers of cells.

Example 12

This example demonstrates that cells transduced with a vector encoding amurine anti-NY-ESO-1 TCR maintain functionality following expansion ofthe numbers of transduced cells.

PBL from two donors were stimulated and either untransduced ortransduced with vectors encoding GFP or the 1G4-LY, mESOαβ, or mESOβαTCR as described in Example 9. The numbers of transduced cells wereexpanded as described in Example 11. Transduced expanded PBL werecultured alone (media) or co-cultured with one of the various tumor celllines listed in Table 14 below or with T2 cells pulsed with SSX peptide,no peptide (T2), or one of the various concentrations of NY-ESO-1₁₅₇₋₁₆₅peptide listed in Table 15 below. IFNγ secretion was measured by ELISAnine days after the second stimulation (S2D9). The results are shown inTables 14 and 15 (IFNγ pg/ml; Dilution 1:10).

TABLE 14 % td COS-A2- H1299- COAS-A2- efficiency Media 888 938 gp100624.38 A2 A375 ESO 1300 Patient 1 Untransduced (UT) N/A 282 181 91 515 78  128  779 —  91 GFP 88 95 87 44 216  44  48  783 —  53 1G4-LY TCR 76208 184 105 260 8233 12848  6454 — 2786 mESOαβ TCR 82 129 183 111 1218132 11976  6152 — 3035 mESOβα TCR 82 92 194 130 148 9104 11381  6226 —2654 Patient 2 (UT) N/A 297 276 195 231  151  144  353 —  167 GFP37 42393 380 205 272  146  186  465 —  168 1G4-LY TCR 37 102 174 53 133 45715984 2420 — 2066 mESOαβ TCR 62 125 397 362 170 7382 9590 4681 — 3439mESOβα TCR 62 137 323 370 130 6345 8250 4236 — 3054

TABLE 15 % td efficiency SSX T2 1 μg/ml 100 ng/ml 10 ng/ml 1 ng/ml 0.1ng/ml Patient 1 Untransduced (UT) NA 365 479  123  113  168  168  231GFP 88 0 30   0   0   0   0   0 1G4 TCR 59 179 275 26446 19424 135306988 1277 1G4-LY TCR 76 185 217 30639 21553 16246 7261 1328 mESOαβ TCR82 511 516 26427 22062 16950 8601 1873 mESOβα TCR 82 399 384 27813 2305216872 8076 2174 Patient 2 Untransduced (UT) NA 976 1281  886  616  680 782  764 GFP 42 660 912  505  697  726  591  763 1G4 TCR 50 279 36419505 12698  7350 3553  747 1G4-LY TCR 37 150 110 19416 10632  6799 2495 662 mESOαβ TCR 62 386 460 19960 13887  9970 4488 1025 mESOβα TCR 62 379611 16434 12708  9646 5266 1177

Functionality of expanded transduced cells was also evaluated bychromium release assay. Expanded transduced cells (effector cells) wereco-cultured with target melanoma cells 624.38 cells (Table 16A) or A375cells (Table 16B) at various effector:target (E:T) cell ratios, and thepercentage of target cells lysed was measured. The results are shown inTables 16A and 16B (percentage of target cells lysed).

TABLE 16A E:T Ratio 1G4-LY TCR mESOα/β TCR mESOβ/α TCR GFP 40:1 71.069.0 68 20.0 13:1 59.0 64.0 62 13.0  4:1 54.0 73.0 46 23.0 1.5:1  42.040.0 39 2.0

TABLE 16B E:T Ratio IG4-LY TCR mESO α/β mESO β/α GFP 40:1 43.0 49.0 5121.0 13:1 43.0 47.0 50 16.0  4:1 36.0 28.0 32 8.0 1.5:1  29.0 31.0 326.0

As shown in Tables 14, 15, 16A, and 16B, cells transduced with a vectorencoding a murine anti-NY-ESO-1 TCR maintained functionality followingexpansion of the numbers of transduced cells.

Example 13

This example demonstrates a method of producing packaging cell clonesfor the production of mESOβα TCR for potential clinical application.

DNA for the mESOβα TCR vector was used to produce retroviral vectorpackaging cell clones under conditions required for potential clinicalapplication. Supernatant from six PG13 producer cell clones was used totransduce PBL. FACS analysis of transduced PBL using the anti-mouseTCR-β chain antibody revealed that each clone produced virus thatmediated positive TCR transduction (Table 17). To assess the specificrecognition of tumor cells, the mTCR engineered PBL from each PG13producer cell clone were co-cultured with a panel of HLA-A*0201⁺ andHLA-A*0201⁻ melanoma and lung tumor derived cell lines (Table 17).IFN-gamma was measured by ELISA. A comparison of the six mTCR PG13producer clones showed that T cells transduced with Clone C1 releasedhigh levels of IFN-γ in response to HLA-A*0201⁺/NY-ESO-1⁺ tumor celltarget H1299-A2 and demonstrated the highest transduction efficiency(Table 17). These responses were specific as background levels of IFN-γwere released in response to NY-ESO-1⁺/HLA-A*0201⁻ cell lines andNY-ESO-1⁻/HLA-A*0201⁺ cell lines by each clone (FIG. 2). Based on thisanalysis, Clone C1 was selected for the production of a master cell bankfor subsequent production of good manufacturing practice (GMP)retroviral supernatant.

TABLE 17 IFN-γ pg/ml Clone % mTCRβ media 888 H1299A2 624.38 A375 UT 4122 1 0 0 146 B2 30 39 46 2923 670 382 C1 63 0 0 7529 942 257 C12 42 100 3332 661 351 D8 36 64 90 5773 675 439 F2 47 31 38 5533 579 488 H4 4434 38 7185 531 459

Example 14

This example demonstrates the transduction efficiency of cellstransduced with a mESOβα TCR using a retroviral supernatant from thepackaging cell clone of Example 13.

To compare the respective NY-ESO-1 TCRs (murine, or mTCR, versus human,or hTCR (1G4-LY TCR)), FACS analysis of PBL transduced with retroviralsupernatant from packaging cell clones using the anti-mouse TCR-Vβ chainand the anti-Vβ13.1 antibodies was performed after one stimulation withOKT3 and following a second large-scale expansion using the rapidexpansion protocol (REP) (Table 18). Results demonstrated that both themTCR and the hTCR had equivalent percentages of transduction afterstimulation, with the mTCR having equal to or greater levels oftransduction after REP (Table 18).

TABLE 18 % TCR Donor H Donor E After After After one expanding After oneexpanding stimulation numbers of stimulation numbers of with OKT3 cellstwice with OKT3 cells twice UT 4 13 4 8 1G4-LY TCR 52 56 48 44 mESOβαTCR 56 61 46 66

Example 15

This example demonstrates the reactivity of cells transduced with amESOβα TCR using a retroviral supernatant from the packaging cell cloneof Example 13.

The recognition of each TCR was evaluated by subjecting the mTCR and thehTCR transduced T cells to co-culture with NY-ESO-1 peptide-pulsed T2cells. Both the mTCR and the hTCR specifically secreted IFN-γ uponencounter with the antigenic peptide in a dose-dependent manner afterone stimulation with OKT3 and after REP (Table 19). After onestimulation, both the mTCR and the hTCR recognized T2 cells pulsed withas little as 0.1 ng/mL indicating that both mTCRs are relatively highavidity receptors. Following the expansion of the numbers of cells, themTCR released higher levels of IFN-γ compared to the hTCR vectortransduced T cells at each concentration of peptide (Table 19).Co-culture of PBL expressing NY-ESO-1 mTCR or NY-ESO-1 hTCR with controlT2 cells that were not pulsed with any peptide produced backgroundlevels of IFN-γ.

TABLE 19 T2 cells peptide without concentration peptide 0.1 ng/μl 1ng/μl 10 ng/μl 100 ng/μl IFN-γ pg/ml Donor H (after stimulation withOKT3) UT 400 380 329 350 285 GFP 633 455 424 410 412 1G4-LY TCR 12591400 1710 3016 3775 mESOβα TCR 1070 1316 1660 3091 3744 IFN-γ pg/mlDonor H (after expanding numbers of cells) UT 34 47 57 22 28 GFP 34 4757 22 28 1G4-LY TCR 24 89 512 2974 4341 mESOβα TCR 174 229 1456 663310683

To assess the specific recognition of tumor cells, the mTCR engineeredPBL were co-cultured with a panel of HLA-A*0201⁺ and HLA-A*0201⁻melanoma and lung tumor derived cell lines. Specific release of IFN-γwas observed when both the mTCR engineered PBL and the hTCR wereco-cultured with HLA-A*0201⁺/NY-ESO-1⁺ cell lines but notHLA-A*0201⁻/NY-ESO-1⁺ or HLA-A*0201⁺/NY-ESO-1⁻ cell lines (Table 20(representative experiments shown)).

TABLE 20 H1299-A2 624.38 1300 938 Patient H Untransduced (UT) 86 93 1180 GFP 83 81 91 0 1G4-LY TCR 14549 6171 1507 0 mESOβα TCR 8877 4248 13260 Patient E Untransduced (UT) 64 72 81 0 GFP 70 72 63 0 1G4-LY TCR 74832149 452 0 mESOβα TCR 10646 3548 986 0

Example 16

This example demonstrates specific lysis of melanoma cells by cellstransduced with a mESOβα TCR using a retroviral supernatant from thepackaging cell clone of Example 13.

The specific lysis of melanoma cell lines by the mTCR and the hTCR werealso compared. The ability of the transduced PBL to lyseHLA-A*0201⁺/NY-ESO-1⁺ tumor cells was measured using a CYTOTOX-GLObioluminescence assay (Promega, Madison, Wis.). This assay utilizes aluminogenic peptide substrate, the AAF-GLO substrate, to measuredead-cell protease activity, which is released from cells that have lostmembrane integrity, resulting in the generation of a “glow-type”luminescent signal that is proportional to the number of dead cells inthe sample. The AAF-GLO substrate cannot cross the intact membrane oflive cells and does not generate any appreciable signal from thelive-cell population. In these assays, TCR engineered PBL wereco-incubated with increasing ratios of target cells (E:T) in AIM-Vmedium in 96-well U-bottom plates at 37° C. for 4 hours (hr.) Lysis wasmeasured by bioluminescence release in the medium: percent specificlysis=[specific release−(spontaneous effector release+spontaneous targetrelease)]/total target release−spontaneous target release×100%, averageof quadruplicate samples. Little or no cell lysis is measured as anegative value.

As shown in Table 21, both mTCR and hTCR transduced PBL demonstratedsimilar lytic activity against melanoma NY-ESO-1⁺/HLA-A*0201⁺ tumor cellline 624.38mel. There was little or no lysis of HLA-A*0201⁻ cell line938 mel, and the GFP transduced PBL showed no reactivity against any ofthe target cells (Table 21).

TABLE 21 Effector:target ratio 10:1 30:1 60:1 Positive Target: 624.38cells GFP −9 −3 −2 1G4-LY TCR 29 59 58 mESOβα TCR 31 59 60 NegativeTarget: 938 cells GFP −19 −22 −34 1G4-LY TCR −13 −11 −10 mESOβα TCR −13−10 −10

Example 17

This example demonstrates the anti-tumor activity of cells transducedwith a mESOβα TCR or human TCR using a retroviral supernatant from thepackaging cell clone of Example 13.

The anti-tumor activity of CD4+ T lymphocytes transduced with the mTCRand the hTCR was also investigated. NY-ESO-1 hTCR and NY-ESO-1 mTCRtransduced PBL were enriched with CD4+ magnetic beads, then co-culturedfor 16 hours with a panel of HLA-A*0201⁺ and HLA-A*0201⁻ melanoma andlung tumor derived cell lines. CD4+ T lymphocytes transduced with boththe mTCR and the hTCR had specific release of IFN-γ when co-culturedwith HLA-A*0201⁺/NY-ESO-1⁺ cell lines but not HLA-A*0201⁻/NY-ESO-1⁺ celllines (Table 22).

TABLE 22 H1299A2 624.38 938 IFN-γ pg/ml (Donor I) Untransduced 0 31 01G4-LY TCR 22684 7020 0 mESOβα TCR 21376 3754 41 IFN-γ pg/ml (Donor J)Untransduced 1158 0 0 1G4-LY TCR 22786 2594 0 mESOβα TCR 22331 481 14

Example 18

This example demonstrates the specific recognition of different tumorhistologies by cells transduced with a mESOβα TCR using a retroviralsupernatant from the packaging cell clone of Example 13.

To assess the specific recognition of various tumor histologies,NY-ESO-1 mTCR transduced PBL were co-cultured with differentHLA-A*0201⁺/NY-ESO-1⁺ cell lines derived from melanoma (A375), non-smallcell lung cancer (H1299-A2), neuroblastoma (SKN AS-A2), breast cancer(MDA-435S-A2), and osteosarcoma (Saos2). Specific release of IFN-γ wasobserved (Table 23).

TABLE 23 IFN-γ pg/ml Untransduced mESOβα TCR A375 0 5710 H1299-A2 13221222 MDA-435S-A2 1181 3057 SKN AS-A2 1417 5097 Saos2 117 12092

Example 19

This example demonstrates the recognition of DAC-treated tumor cells byPBL transduced with a mESOβα TCR.

Increasing concentrations of the DNA-demethylating agent5-aza-2′-deoxycytidine (decitabine; DAC) induces expression of variouscancer testis antigens in lung cancer cells (Rao et al., Ther. Tar. andChem. Bio., 71: 4192-4204 (2011)). Without being bound to a particulartheory or mechanism, it is believed that DAC may, potentially,up-regulate NY-ESO-1 expression in cancer cells, which may enhance theability of the TCRs to recognize NY-ESO-1.

NY-ESO-1 mTCR transduced or untransduced PBL were co-cultured for 16hours with the tumor target cell lines of different histologies (shownin Tables 24A-24B) that had been exposed to DAC at the concentrationsshown in Tables 24A-24B for 72 hours. Interferon-gamma levels weremeasured. The results are shown in Table 24A-24B.

TABLE 24A Prostate Cancer (pC3A2 cells) DAC concentration IFN-γ (pg/ml)(mM/L) Untransduced mESOβα TCR from Clone C1 Untreated 159 336 0.1 2891566 0.5 188 1766 1.0 282 1912 10 361 1520

TABLE 24B Colorectal Cancer (SW480 cells) DAC concentration IFN-γ(pg/ml) (mM/L) Untransduced mESOβα TCR from Clone C1 Untreated 118 1350.1 141 196 0.5 169 239 1.0 98 255 10 80 388

As shown in Tables 24A and 24B, the PBL transduced with mESOβα TCRdemonstrated higher reactivity toward DAC-treated target prostate cancerand colorectal cancer, respectively, as compared to untreated targetcells.

Example 20

This example demonstrates the recognition of T2 cells pulsed withalanine-substituted NY-ESO-1 peptides by PBL transduced with the mESOβαTCR or human TCR.

Untransduced human PBL or human PBL transduced with mTCR (mESOβα fromclone C1), hTCR (1G4-LY TCR), or green fluorescent protein (GFP) wereco-cultured for 16 hours with untreated T2 cells or T2 cells that werepreviously pulsed with different concentrations of peptide as shown inTables 25A and 25B. Interferon gamma was measured. The results are shownin Table 25A and 25B. As shown in Tables 25A and 25B, the mTCRrecognizes SEQ ID NO: 24 while the hTCR does not. In addition, the hTCRrecognizes SEQ ID NO: 27 but the mTCR does not.

TABLE 25A (Donor K) pulsed peptide IFN-γ (pg/ml) (ng/μL) UntransducedGFP 1G4-LY TCR mESOβα untreated — 943 358  641  443 T2 Cells SLLMWITQC10   0   0 3759 5741 (SEQ ID NO: 2) SLLMWITQC  1   0   0  678 2111(SEQ ID NO: 2) MART 10   0   0    0    0 MART  1   0   0    0    0 SL AMWITQC 10   0   0 3271 6287 (SEQ ID NO: 23) SL A MWITQC  1   0   0 17852421 (SEQ ID NO: 23) SLL A WITQC 10   0   0    0 2701 (SEQ ID NO: 24)SLL A WITQC  1   0   0    0 3172 (SEQ ID NO: 24) SLLM A ITQC 10   0   0   0    0 (SEQ ID NO: 25) SLLM A ITQC  1   0   0    0    0(SEQ ID NO: 25) SLLMW A TQC 10   0   0 1114 1194 (SEQ ID NO: 26) SLLMW ATQC  1   0   0  884  921 (SEQ ID NO: 26) SLLMWI A QC 10   0   0 5672   0 (SEQ ID NO: 27) SLLMWI A QC  1   0   0  457    0 (SEQ ID NO: 27)SLLMWIT A C 10   0   0    0    0 (SEQ ID NO: 28) SLLMWIT A C  1   0   0   0    0 (SEQ ID NO: 28)

TABLE 25B (Donor L) pulsed peptide IFN-γ (pg/ml) (ng/μL) UntransducedGFP 1G4-LY TCR mESOβα untreated — 162 126  131  188 T2 Cells SLLMWITQC10 124 120  754 1168 (SEQ ID NO: 2) SLLMWITQC  1 199 112  184  273(SEQ ID NO: 2) MART 10 230 136  123  102 MART  1 155 152  112  108 SL AMWITQC 10 145 168 1383 1541 (SEQ ID NO: 23) SL A MWITQC  1 120 139  370 505 (SEQ ID NO: 23) SLL A WITQC 10 128 121  185  616 (SEQ ID NO: 24)SLL A WITQC  1 123 111  225 1000 (SEQ ID NO: 24) SLLM A ITQC 10 183 163 294  137 (SEQ ID NO: 25) SLLM A ITQC  1 119 112  148  214(SEQ ID NO: 25) SLLMW A TQC 10  96  80  297  375 (SEQ ID NO: 26) SLLMW ATQC  1  74  70  220  280 (SEQ ID NO: 26) SLLMWI A QC 10  78  95 1026  76 (SEQ ID NO: 27) SLLMWI A QC  1 123  72  208   86 (SEQ ID NO: 27)SLLMWIT A C 10  78  83   97   76 (SEQ ID NO: 28) SLLMWIT A C  1  85  75  82   74 (SEQ ID NO: 28)

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The term “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of specifically binding a T-cell receptor (TCR) to a cancer cell in a mammal, the method comprising administering an isolated host cell or a population thereof to the mammal in an amount effective to treat specifically bind the TCR to the cancer cell in the mammal, wherein the isolated host cell expresses the TCR, wherein the TCR has antigenic specificity for NY-ESO-1 (SEQ ID NO: 1) and comprises a murine variable region, wherein the TCR comprises the amino acid sequences of SEQ ID NOs: 3-8, and wherein the cancer cell is a NY-ESO-1 positive cancer cell.
 2. The method according to claim 1, comprising administering the population of isolated host cells to the mammal in an amount effective to specifically bind the TCR to the cancer cell in the mammal.
 3. The method of claim 1, wherein the cancer cell is a melanoma cell.
 4. The method of claim 1, wherein the cancer cell is a breast cancer cell.
 5. The method of claim 1, wherein the cancer cell is a lung cancer cell.
 6. The method of claim 1, wherein the cancer cell is a prostate cancer cell.
 7. The method of claim 1, wherein the cancer cell is a thyroid cancer cell.
 8. The method of claim 1, wherein the cancer cell is an ovarian cancer cell.
 9. The method of claim 1, wherein the cancer cell is a synovial cell sarcoma cell.
 10. The method of claim 1, wherein the TCR comprises the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO:
 10. 11. The method of claim 1, wherein the TCR comprises the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO:
 12. 